Platelet-activating factor acetylhydrolase

ABSTRACT

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

This is a continuation of U.S. patent application Ser. No. 08/910,041,filed Aug. 12, 1997 which is a continuation-in-part of U.S. patentapplication Ser. No. 08/483,232 filed Jun. 7, 1995 now U.S. Pat. No.5,656,431, which in turn is a continuation-in-part of U.S. patentapplication Ser. No. 08/318,905 filed Oct. 6, 1994, now U.S. Pat. No.5,641,669, which in turn is a continuation-in-part of U.S. patentapplication Ser. No. 08/133,803 filed Oct. 6, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to platelet-activating factoracetylhydrolase and more specifically to novel purified and isolatedpolynucleotides encoding human plasma platelet-activating factoracetylhydrolase, to the platelet-activating factor acetylhydrolaseproducts encoded by the polynucleotides, to materials and methods forthe recombinant production of platelet-activating factor acetylhydrolaseproducts and to antibody substances specific for platelet-activatingfactor acetylhydrolase.

BACKGROUND

Platelet-activating factor (PAF) is a biologically active phospholipidsynthesized by various cell types. In vivo and at normal concentrationsof 10⁻¹⁰ to 10⁻⁹ M, PAF activates target cells such as platelets andneutrophils by binding to specific G protein-coupled cell surfacereceptors [Venable et al., J. Lipid Res., 34: 691-701 (1993)]. PAF hasthe structure 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. Foroptimal biological activity, the sn-1 position of the PAF glycerolbackbone must be in an ether linkage with a fatty alcohol and the sn-3position must have a phosphocholine head group.

PAF functions in normal physiological processes (e.g., inflammation,hemostasis and parturition) and is implicated in pathologicalinflammatory responses (e.g., asthma, anaphylaxis, septic shock andarthritis) [Venable et al., supra, and Lindsberg et al., Ann. Neurol.,30: 117-129 (1991)]. The likelihood of PAF involvement in pathologicalresponses has prompted attempts to modulate the activity of PAF and themajor focus of these attempts has been the development of antagonists ofPAF activity which interfere with binding of PAF to cell surfacereceptors. See, for example, Heuer et al., Clin. Exp. Allergy, 22:980-983 (1992).

The synthesis and secretion of PAF as well as its degradation andclearance appear to be tightly controlled. To the extent thatpathological inflammatory actions of PAF result from a failure of PAFregulatory mechanisms giving rise to excessive production, inappropriateproduction or lack of degradation, an alternative means of modulatingthe activity of PAF would involve mimicing or augmenting the naturalprocess by which resolution of inflammation occurs. Macrophages[Stafforini et al., J. Biol. Chem., 265(17): 9682-9687 (1990)],hepatocytes and the human hepatoma cell line HepG2 [Satoh et al., J.Clin. Invest., 87: 476-481 (1991) and Tarbet et al., J. Biol. Chem.,266(25): 16667-16673 (1991)] have been reported to release an enzymaticactivity, PAF acetylhydrolase (PAF-AH), that inactivates PAF. Inaddition to inactivating PAF, PAF-AH also inactivates oxidativelyfragmented phospholipids such as products of the arachidonic acidcascade that mediate inflammation. See, Stremler et al., J. Biol. Chem.,266(17): 11095-11103 (1991). The inactivation of PAF by PAF-AH occursprimarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AHmetabolizes oxidatively fragmented phospholipids by removing sn-2 acylgroups. Two types of PAF-AH have been identified: cytoplasmic formsfound in a variety of cell types and tissues such as endothelial cellsand erythrocytes, and an extracellular form found in plasma and serum.Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF andthis substrate specificity allows the enzyme to circulate in vivo in afully active state without adverse effects. The plasma PAF-AH appears toaccount for all of the PAF degradation in human blood ex vivo[Stafforini et al., J. Biol. Chem., 262(9): 4223-4230 (1987)].

While the cytoplasmic and plasma forms of PAF-AH appear to haveidentical substrate specificity, plasma PAF-AH has biochemicalcharacteristics which distinguish it from cytoplasmic PAF-AH and fromother characterized lipases. Specifically, plasma PAF-AH is associatedwith lipoprotein particles, is inhibited by diisopropyl fluorophosphate,is not affected by calcium ions, is relatively insensitive toproteolysis, and has an apparent molecular weight of 43,000 daltons.See, Stafforini et al. (1987), supra. The same Stafforini et al. articledescribes a procedure for partial purification of PAF-AH from humanplasma and the amino acid composition of the plasma material obtained byuse of the procedure. Cytoplasmic PAF-AH has been purified fromerythrocytes as reported in Stafforini et al., J. Biol. Chem., 268(6):3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AHare also described in the article. Hattori et al., J. Biol. Chem.,268(25): 18748-18753 (1993) describes the purification of cytoplasmicPAF-AH from bovine brain. Subsequent to filing of the parent applicationhereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH waspublished in Hattori et al., J. Biol. Chem., 269(237): 23150-23155(1994). On Jan. 5, 1995, three months after the filing date of theparent application hereto, a nucleotide sequence for a lipoproteinassociated phospholipase A₂ (Lp-PLA₂) was published in SmithklineBeecham PLC Patent Cooperation Treaty (PCT) International PublicationNo. WO 95/00649. The nucleotide sequence of the Lp-PLA₂ differs at oneposition when compared to the nucleotide sequence of the PAF-AH of thepresent invention. The nucleotide difference (corresponding to position1297 of SEQ ID NO: 7) results in an amino acid difference between theenzymes encoded by the polynucleotides. The amino acid at position 379of SEQ ID NO: 8 is a valine while the amino acid at the correspondingposition in Lp-PLA₂ is an alanine. In addition, the nucleotide sequenceof the PAF-AH of the present invention includes 124 bases at the 5' endand twenty bases at the 3' end not present in the Lp-PLA₂ sequence.Three months later, on Apr. 10, 1995, a Lp-PLA₂ sequence was depositedin GenBank under Accession No. U24577 which differs at eleven positionswhen compared to the nucleotide sequence of the PAF-AH of the presentinvention. The nucleotide differences (corresponding to position 79, 81,84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO: 7)results in four amino acid differences between the enzymes encoded bythe polynucleotides. The amino acids at positions 249, 250, 274 and 389of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine,respectively, while the respective amino acid at the correspondingpositions in the GenBank sequence are isoleucine, arginine, leucine andserine.

The recombinant production of PAF-AH would make possible the use ofexogenous PAF-AH to mimic or augment normal processes of resolution ofinflammation in vivo. The administration of PAF-AH would provide aphysiological advantage over administration of PAF receptor antagonistsbecause PAF-AH is a product normally found in plasma. Moreover, becausePAF receptor antagonists which are structurally related to PAF inhibitnative PAF-AH activity, the desirable metabolism of PAF and ofoxidatively fragmented phospholipids is thereby prevented. Thus, theinhibition of PAF-AH activity by PAF receptor antagonists counteractsthe competitive blockade of the PAF receptor by the antagonists. See,Stremler et al., supra. In addition, in locations of acute inflammation,for example, the release of oxidants results in inactivation of thenative PAF-AH enzyme in turn resulting in elevated local levels of PAFand PAF-like compounds which would compete with any exogenouslyadministed PAF receptor antagonist for binding to the PAF receptor. Incontrast, treatment with recombinant PAF-AH would augment endogenousPAF-AH activity and compensate for any inactivated endogenous enzyme.

There thus exists a need in the art to identify and isolatepolynucleotide sequences encoding human plasma PAF-AH, to developmaterials and methods useful for the recombinant production of PAF-AHand to generate reagents for the detection of PAF-AH in plasma.

SUMMARY OF THE INVENTION

The present invention provides novel purified and isolatedpolynucleotides (i.e., DNA and RNA both sense and antisense strands)encoding human plasma PAF-AH or enzymatically active fragments thereof.Preferred DNA sequences of the invention include genomic and cDNAsequences as well as wholly or partially chemically synthesized DNAsequences. The DNA sequence encoding PAF-AH that is set out in SEQ IDNO: 7 and DNA sequences which hybridize to the noncoding strand thereofunder standard stringent conditions or which would hybridize but for theredundancy of the genetic code, are contemplated by the invention. Alsocontemplated by the invention are biological replicas (i.e., copies ofisolated DNA sequences made in vivo or in vitro) of DNA sequences of theinvention. Autonomously replicating recombinant constructions such asplasmid and viral DNA vectors incorporating PAF-AH sequences andespecially vectors wherein DNA encoding PAF-AH is operatively linked toan endogenous or exogenous expression control DNA sequence and atranscription terminator are also provided.

According to another aspect of the invention, procaryotic or eucaryotichost cells are stably transformed with DNA sequences of the invention ina manner allowing the desired PAF-AH to be expressed therein. Host cellsexpressing PAF-AH products can serve a variety of useful purposes. Suchcells constitute a valuable source of immunogen for the development ofantibody substances specifically immunoreactive with PAF-AH. Host cellsof the invention are conspicuously useful in methods for the large scaleproduction of PAF-AH wherein the cells are grown in a suitable culturemedium and the desired polypeptide products are isolated from the cellsor from the medium in which the cells are grown by, for example,immunoaffinity purification.

A non-immunological method contemplated by the invention for purifyingPAF-AH from plasma includes the following steps: (a) isolating lowdensity lipoprotein particles; (b) solubilizing said low densitylipoprotein particles in a buffer comprising 10 mM CHAPS to generate afirst PAF-AH enzyme solution; (c) applying said first PAF-AH enzymesolution to a DEAE anion exchange column; (d) washing said DEAE anionexchange column using an approximately pH 7.5 buffer comprising 1 mMCHAPS; (e) eluting PAF-AH enzyme from said DEAE anion exchange column infractions using approximately pH 7.5 buffers comprising a gradient of 0to 0.5 M NaCl; (f) pooling fractions eluted from said DEAE anionexchange column having PAF-AH enzymatic activity; (g) adjusting saidpooled, active fractions from said DEAE anion exchange column to 10 mMCHAPS to generate a second PAF-AH enzyme solution; (h) applying saidsecond PAF-AH enzyme solution to a blue dye ligand affinity column; (i)eluting PAF-AH enzyme from said blue dye ligand affinity column using abuffer comprising 10 mM CHAPS and a chaotropic salt; (j) applying theeluate from said blue dye ligand affinity column to a Cu ligand affinitycolumn; (k) eluting PAF-AH enzyme from said Cu ligand affinity columnusing a buffer comprising 10 mM CHAPS and imidazole; (l) subjecting theeluate from said Cu ligand affinity column to SDS-PAGE; and (m)isolating the approximately 44 kDa PAF-AH enzyme from theSDS-polyacrylamide gel. Preferably, the buffer of step (b) is 25 mMTris-HCl, 10 mM CHAPS, pH 7.5; the buffer of step (d) is 25 mM Tris-HCl,1 mM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column;the buffer of step (i) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5M KSCN, pH7.5; the column of step (j) is a Cu Chelating Sepharose column; and thebuffer of step (k) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5M NaCl, 50 mMimidazole at a pH in a range of about pH 7.5-8.0.

A method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli producing PAF-AH includes thesteps of: (a) preparing a centrifugation supernatant from lysed E. coliproducing PAF-AH enzyme; (b) applying said centrifugation supernatant toa blue dye ligand affinity column; (c) eluting PAF-AH enzyme from saidblue dye ligand affinity column using a buffer comprising 10 mM CHAPSand a chaotropic salt; (d) applying said eluate from said blue dyeligand affinity column to a Cu ligand affinity column; and (e) elutingPAF-AH enzyme from said Cu ligand affinity column using a buffercomprising 10 mM CHAPS and imidazole. Preferably, the column of step (b)is a Blue Sepharose Fast Flow column; the buffer of step (c) is 25 mMTris-HCl, 10 mM CHAPS, 0.5M KSCN, pH 7.5; the column of step (d) is a CuChelating Sepharose column; and the buffer of step (e) is 25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, 100 mM imidazole, pH 7.5.

Another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli producing PAF-AH includes thesteps of: (a) preparing a centrifugation supernatant from lysed E. coliproducing PAF-AH enzyme; (b) diluting said centrifugation supernatant ina low pH buffer comprising 10 mM CHAPS; (c) applying said dilutedcentrifugation supernatant to a cation exchange column equilibrated atabout pH 7.5; (d) eluting PAF-AH enzyme from said cation exchange columnusing 1M salt; (e) raising the pH of said eluate from said cationexhange column and adjusting the salt concentration of said eluate toabout 0.5M salt; (f) applying said adjusted eluate from said cationexchange column to a blue dye ligand affinity column; (g) eluting PAF-AHenzyme from said blue dye ligand affinity column using a buffercomprising about 2M to about 3M salt; and (h) dialyzing said eluate fromsaid blue dye ligand affinity column using a buffer comprising about0.1% Tween. Preferably, the buffer of step (b) is 25 mM MES, 10 mMCHAPS, 1 mM EDTA, pH 4.9; the column of step (c) is an S sepharosecolumn equilibrated in 25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH5.5; PAF-AH is eluted in step (d) using 1 mM NaCl; the pH of the eluatein step (e) is adjusted to pH 7.5 using 2M Tris base; the column in step(f) is a sepharose column; the buffer in step (g) is 25 mM Tris, 10 mMCHAPS, 3M NaCl, 1 mM EDTA, pH 7.5; and the buffer in step (h) is 25 mMTris, 0.5M NaCl, 0.1% Tween 80, pH 7.5.

Still another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli includes the steps of: (a)preparing an E. coli extract which yields solubilized PAF-AH supernatantafter lysis in a buffer containing CHAPS; (b) dilution of saidsupernatant and application to a anion exchange column equilibrated atabout pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column;(d) applying said adjusted eluate from said anion exchange column to ablue dye ligand affinity column; (e) eluting the said blue dye ligandaffinity column using a buffer comprising 3.0M salt; (f) dilution of theblue dye eluate into a suitable buffer for performing hydroxylapatitechromatography; (g) performing hydroxylapatite chromatography wherewashing and elution is accomplished using buffers (with or withoutCHAPS); (h) diluting said hydroxylapatite eluate to an appropriate saltconcentration for cation exchange chromatography; (i) applying saiddiluted hydroxylapatite eluate to a cation exchange column at a pHranging between approximately 6.0 to 7.0; (j) elution of PAF-AH fromsaid cation exchange column with a suitable formulation buffer, (k)performing cation exchange chromatography in the cold; and (l)formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step (a) above the lysis buffer is 25 mM Tris, 100 mMNaCl, 1 mM EDTA, 20 mM CHAPS, pH 8.0; in step (b) the dilution of thesupernatant for anion exchange chromatography is 3-4 fold into 25 mMTris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 and the column is a Q-Sepharosecolumn equilibrated with 25 mM Tris, 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS,pH 8.0; in step (c) the anion exchange column is eluted using 25 mMTris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0; in step (d) theeluate from step (c) is applied directly onto a blue dye affinitycolumn; in step (e) the column is eluted with 3M NaCl, 10 mM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate forhydroxylapatite chromatography is accomplished by dilution into 10 mMsodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH 6.2; in step (g)hydroxylapatite chromatography is accomplished using a hydroxylapatitecolumn equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mMCHAPS and elution is accomplished using 50 mM sodium phosphate. 100 mMNaCl (with or without) 10 mM CHAPS, pH 7.5; in step (h) dilution of saidhydroxylapatite eluate for cation exchange chromatography isaccomplished by dilution into a buffer ranging in pH from approximately6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step(i) a S Sepharose column is equilibrated with 50 mM sodium phosphate,(with or without) 10 mM CHAPS, pH 6.8; in step (j) elution isaccomplished with a suitable formulation buffer such as potassiumphosphate 50 mM, 12.5 mM aspartic acid, 125 mM NaCl, pH 7.5 containing0.01% Tween-80; and in step (k) cation exchange chromatrography isaccomplished at 2-8° C. Examples of suitable formulation buffers for usein step (l) which stabilize PAF-AH include 50 mM potassium phosphate,12.5 mM Aspartic acid, 125 mM NaCl pH 7.4 (approximately, with andwithout the addition of Tween-80 and or Pluronic F68) or 25 mM potassiumphosphate buffer containing (at least) 125 mM NaCl, 25 mM arginine and0.01% Tween-80 (with or without Pluronic F68 at approximately 0.1 and0.5%).

Yet another method contemplated by the invention for purifyingenzymatically active rPAF-AH products from E. coli includes the stepsof: (a) preparing an E. coli extract which yields solubilized rPAF-AHproduct supernatant after lysis in a buffer containing Triton X-100, (b)dilution of said supernatant and application to an immobilized metalaffinity exchange column equilibrated at about pH 8.0; (c) elutingrPAF-AH product from said immobilized metal affinity exchange columnwith a buffer comprising imidazole; (d) adjusting the salt concentrationand applying said eluate from said immobilized metal affinity column toan hydrophobic interaction column (HIC#1); (e) eluting said HIC#1 byreducing the salt concentration and/or increasing the detergentconcentration; (f) titrating said HIC#1 eluate to a pH of about 6.4; (g)applying said adjusted HIC#1 eluate to a cation exchange column (CEX#1)equilibrated at about pH 6.4; (h) eluting said CEX#1 with concentration?sodium chloride; (i) adjusting said CEX#1 eluate with sodium chloride toa concentration of about 2.0M; (j) applying said adjusted CEX#1 eluateto a hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0and about 2.0M sodium chloride; (k) eluting said HIC#2 hy reducing thesalt concentration and/or increasing the detergent concentration; (1)diluting said HIC#2 eluate and adjusting to a pH of about 6.0; (m)applying said adjusted HIC#2 eluate to a cation exchange column (CEX2)equilibrated at about pH 6.0; (n) eluting the rPAF-AH product from saidCEX#2 with a suitable formulation buffer.

Preferably, in step (a) above the lysis buffer is 90 mM TRIS, 0.125%Triton X-100, 0.6M NaCl, pH 8.0, and lysis is carried out in a highpressure homogenizer; in step (b) the supernatant is diluted intoequilibration buffer (20 mM TRIS, 0.5M NaCl, 0.1% Triton X-100, pH 8.0),a zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia,Uppsala, Sweden) is charged, equilibrated with equilibration buffer,loaded with the diluted supernatant, and washed with 20 mM TRIS, 0.5MNaCl, 4M urea, 0.1% Triton X-100, pH 8.0, followed by washing with 20 mMTRIS, 0.5M NaCl, 0.02% Triton X-100, pH 8.0; in step (c) elution isaccomplished with 20 mM Tris, 50 mM imidazole, 0.02% Triton X-100, pH8.0; in step (d) the eluate is adjusted to 1 mM EDTA and 2M NaCl, aPhenyl Sepharose 6 Fast Flow (Pharmacia) is equilibrated withequilibration buffer (2.0M NaCl, 25 mM Tris, 0.02% Triton X-100, pH8.0), loaded with the adjusted eluate from step (c) at room temperature,washed with equilibration buffer, and washed with 25 mM NaPO₄, 0.02%Triton X-100, pH6.5 at a flow rate of 30 cm/hr; in step (e) elution isaccomplished with 25 mM NaPO₄, 3% Triton X-100, pH 6.5; in step (g) aMacro-Prep High S Column (Bio-Rad Labs, Richmond, Calif.) isequilibrated with equilibration buffer (20 mM NaPO₄, 0.02% Triton X-100,pH 6.4), loaded with the adjusted eluate from step (f), washed withequilibration buffer, and washed with 25 mM Tris, 0.02% Triton X-100, pH8.0; in step (h) elution is accomplished with 25 mM Tris, 0.02% TritonX-100, 1.3M NaCl, pH 8.0; in step (j) a Bakerbond Wide Pore Hi-Propyl C₃(Baker, Phillipsburg, N.J.) is equilibrated with equilibration buffer(2.0M NaCl, 25 mM Tris, 0.02% Triton X-100, pH 8.0), loaded withadjusted eluate from step (i) at room temperature, washed withequilibration buffer, and washed with 25 mM Tris, 0.02% Triton X-100, pH8.0 at 30 cm/hr; in step (k) elution is accomplished with 10 mM Tris,3.0% Triton X-100, pH 8.0; in step (l) dilution is into equilibrationbuffer (20 mM succinate, 0.1% PLURONIC F68, pH 6.0); in step (m) a SPSepharose Fast Flow (Pharmacia) column is equilibrated with theequilibration buffer of step (l), loaded with eluate from step (l), andwashed with equilibration buffer; and in step (n) elution isaccomplished with 50 mM NaPO₄, 0.7M NaCl, 0.1% PLURONIC F68, 0.02% TWEEN80, pH 7.5.

PAF-AH products may be obtained as isolates from natural cell sources ormay be chemically synthesized, but are preferably produced byrecombinant procedures involving procaryotic or eucaryotic host cells ofthe invention. PAF-AH products having part or all of the amino acidsequence set out in SEQ ID NO: 8 are contemplated. Specificallycontemplated are fragments lacking up to the first twelve N-terminalamino acids of the mature human PAF-AH amino acid sequence set out inSEQ ID NO: 8, particularly those having Met₄₆, Ala₄₇ or Ala₄₈ of SEQ IDNO: 8 as the initial N-terminal amino acid. Also contemplated arefragments thereof lacking up to thirty C-terminal amino acids of theamino acid sequence of SEQ ID NO: 8, particularly those having Ile₄₂₉and Leu₄₃₁ as the C-terminal residue. Further contemplated are variantsof PAF-AH or PAF-AH or which have an amino acid replacement in thesequence of SEQ ID NO. 8 selected from the group consisting of S 108 A,S 273 A, D 286 A, D 286 N, D 296 A, D 304 A, D 338 A, H 351 A, H 395 A,H 399 A, C 67 S, C 229 S, C 291 S, C 334 S, C 407 S, D 286 A, D 286 Nand D 304 A. As noted above, polynucleotides (including DNA) encodingsuch fragments or variant fragments are provided by the invention, aswell as methods of recombinantly producing such fragments or variants bygrowing host cells comprising such DNA. Presently preferred PAF-AHproducts include the prokaryotic polypeptide expression products of DNAencoding amino acid residues Met₄₆ through Asn₄₄₁ of SEQ ID NO: 8,designated rPH.2, and the prokaryotic polypeptide expression products ofDNA encoding amino acid residues Met₄₆ through Ile₄₂₉ of SEQ ID NO: 8,designated rPH.9. Both the rPH.2 and rPH.9 products display lessamino-terminal heterogeneity than, for example, the correspondingprokaryotic expression products of DNA encoding the full mature sequenceof PAF-AH preceded by a translation initiation codon. Moreover, therPH.9 product displays greater carboxy terminal homogeneity(consistency). The use of mammalian host cells is expected to providefor such post-translational modifications (e.g., myristolation,glycosylation, truncation, lipidation and tyrosine, serine or threoninephosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. PAF-AH products ofthe invention may be full length polypeptides, fragments or variants.Variants may comprise PAF-AH analogs wherein one or more of thespecified (i.e., naturally encoded) amino acids is deleted or replacedor wherein one or more nonspecified amino acids are added: (1) withoutloss of one or more of the enzymatic activities or immunologicalcharacteristics specific to PAF-AH; or (2) with specific disablement ofa particular biological activity of PAF-AH. Proteins or other moleculesthat bind to PAF-AH may be used to modulate its activity.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, CDR-grafted antibodies and the like) and otherbinding proteins specific for PAF-AH. Specifically illustrating bindingproteins of the invention are the monoclonal antibodies produced byhybridomas 90G11D and 90F2D which were deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 onSep. 30, 1994 and were respectively assigned Accession Nos. HB 11724 andHB 11725. Also illustrating binding proteins of the invention is themonoclonal antibody produced by hybridoma 143A which was deposited withthe ATCC on Jun. 1, 1995 and assigned Accession No. HB 11900. Proteinsor other molecules (e.g., lipids or small molecules) which specificallybind to PAF-AH can be identified using PAF-AH isolated from plasma,recombinant PAF-AH, PAF-AH variants or cells expressing such products.Binding proteins are useful, in turn, in compositions for immunizationas well as for purifying PAF-AH, and are useful for detection orquantification of PAF-AH in fluid and tissue samples by knownimmunological procedures. Anti-idiotypic antibodies specific forPAF-AH-specific antibody substances are also contemplated.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor PAF-AH makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding PAF-AH and specifying PAF-AH expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention under conditions of stringency standard in the art arelikewise expected to allow the isolation of DNAs encoding allelicvariants of PAF-AH, other structurally related proteins sharing one ormore of the biochemical and/or immunological properties of PAF-AH, andnon-human species proteins homologous to PAF-AH. The DNA sequenceinformation provided by the present invention also makes possible thedevelopment, by homologous recombination or "knockout" strategies [see,e.g., Kapecchi, Science, 244: 1288-1292 (1989)], of rodents that fail toexpress a functional PAF-AH enzyme or that express a variant PAF-AHenzyme. Polynucleotides of the invention when suitably labelled areuseful in hybridization assays to detect the capacity of cells tosynthesize PAF-AH. Polynucleotides of the invention may also be thebasis for diagnostic methods useful for identifying a geneticalteration(s) in the PAF-AH locus that underlies a disease state orstates. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of PAF-AH by thosecells which ordinarily express the same.

Administration of PAF-AH preparations of the invention to mammaliansubjects, especially humans, for the purpose of amelioratingpathological inflammatory conditions is contemplated. Based onimplication of the involvement of PAF in pathological inflammatoryconditions, the administration of PAF-AH is indicated, for example, intreatment of asthma [Miwa et al., J. Clin. Invest., 82: 1983-1991(1988); Hsieh et a., J. Allergy Clin. Immunol., 91: 650-657 (1993); andYamashita et al., Allergy, 49: 60-63 (1994)], anaphylaxis [Venable etal., supra], shock [Venable et al., supra], reperfusion injury andcentral nervous system ischemia [Lindsberg et al. (1991), supra],antigen-induced arthritis [Zarco et al., Clin. Exp. Immunol., 88:318-323 (1992)], atherogenesis [Handley et al., Drug Dev. Res., 7:361-375 (1986)], Crohn's disease [Denizot et al., Digestive Diseases andSciences, 37(3): 432-437 (1992)], ischemic bowel necrosis/necrotizingenterocolitis [Denizot et al., supra and Caplan et al., Acta Paediatr.,Suppl. 396: 11-17 (1994)], ulcerative colitis (Denizot et al., supra),ischemic stroke [Satoh et al., Stroke, 23: 1090-1092 (1992)], ischemicbrain injury [Lindsberg et al., Stroke, 21: 1452-1457 (1990) andLindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzakiet al., Clinica Chimica Acta, 210: 139-144 (1992)], acute pancreatitis[Kald et al., Pancreas, 8(4): 440-442 (1993)], septicemia (Kald et al.,supra), acute post streptococcal glomerulonephritis [Mezzano et al., J.Am. Soc. Nephrol., 4: 235-242 (1993)], pulmonary edema resulting fromIL-2 therapy [Rabinovici et al., J. Clin. Invest., 89: 1669-1673(1992)], allergic inflammation [Watanabe et al., Br. J. Pharmacol., 111:123-130 (1994)], ischemic renal failure [Grino et al., Annals ofInternal Medicine, 121(5): 345-347 (1994); preterm labor [Hoffman etal., Am. J. Obstet. Gynecol., 162(2): 525-528 (1990) and Maki et al.,Proc. Natl. Acad. Sci. USA, 85: 728-732 (1988)]; adult respiratorydistress syndrome [Rabinovici et al., J. Appl. Physiol., 74(4):1791-1802 (1993); Matsumoto et al., Clin. Exp. Pharmacol. Physiol., 19509-515 (1992); and Rodriguez-Roisin et al., J. Clin. Invest., 93:188-194 (1994)]. Also contemplated is the use of PAF-AH preparations totreat human immunodeficiency virus (HIV) infection of the centralnervous system. "Treatment" as used herein includes both prophylacticand therapeutic treatment.

Animal models for many of the foregoing pathological conditions havebeen described in the art. For example, a mouse model for asthma andrhinitis is described in Example 16 herein; a rabbit model for arthritisis described in Zarco et at., supra; rat models for ischemic bowelnecrosis/necrotizing enterocolitis are described in Furukawa et al.,Ped. Res., 34,(2): 237-241 (1993) and Caplan et al., supra; a rabbitmodel for stroke is described in Lindsberg et al., (1990), supra; amouse model for lupus is described in Matsuzaki et al., supra; a ratmodel for acute pancreatitis is described in Kald et al., supra: a ratmodel for pulmonary edema resulting from IL-2 therapy is described inRabinovici et al., supra; a rat model of allergic inflammation isdescribed in Watanabe et al., supra); a canine model of renal allograftis described in Watson et al., Transplantation, 56(4): 1047-1049 (1993);and rat and guinea pig models of adult respiratory distress syndrome arerespectively described in Rabinovici et al., supra. andLellouch-Tubiana, Am. Rev. Respir. Dis., 137: 948-954 (1988).

Specifically contemplated by the invention are PAF-AH compositions foruse in methods for treating a mammal susceptible to or suffering fromPAF-mediated pathological conditions comprising administering PAF-AH tothe mammal in an amount sufficient to supplement endogenous PAF-AHactivity and to inactivate pathological amounts of PAF in the mammal.

Therapeutic/pharmaceutical compositions contemplated by the inventioninclude PAF-AH products and a physiologically acceptable diluent orcarrier and may also include other agents having anti-inflammatoryeffects. Dosage amounts indicated would be sufficient to supplementendogenous PAF-AH activity and to inactivate pathological amounts ofPAF. For general dosage considerations see Remington's PharmaceuticalSciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990). Dosageswill vary between about 0.1 to about 1000 μg PAF-AH/kg body weight.Therapeutic compositions of the invention may be administered by variousroutes depending on the pathological condition to be treated. Forexample, administration may be by intraveneous, subcutaneous, oral,suppository, and/or pulmonary routes.

For pathological conditions of the lung, administration of PAF-AH by thepulmonary route is particularly indicated. Contemplated for use inpulmonary administration are a wide range of delivery devices including,for example, nebulizers, metered dose inhalers, and powder inhalers,which are standard in the art. Delivery of various proteins to the lungsand circulatory system by inhalation of aerosol formulations has beendescribed in Adjei et al., Pharm. Res., 7(6): 565-569 (1990) (leuprolideacetate); Braquet et al. J. Cardio. Pharm. 13(Supp. 5): s. 143-146(1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine,III(3), 206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest.,84: 1145-1146 (1989) (α-1-proteinase inhibitor); Debs et al., J.Immunol. , 140: 3482-3488 (1933) (recombinant gamma interferon and tumornecrosis factor alpha); Patent Cooperation Treaty (PCT) InternationalPublication No. WO 94/20069 published Sep. 15, 1994 (recombinantpegylated granulocyte colony stimulating factor).

BRIEF DESCRIPTION OF THE DRAWING

Numerous other aspects and advantages of the present invention will beapparent upon consideration of the following detailed descriptionthereof, reference being made to the drawing wherein:

FIG. 1 is a photograph of a PVDF membrane containing PAF-AH purifiedfrom human plasma;

FIG. 2 is a graph showing the enzymatic activity of recombinant humanplasma PAF-AH;

FIG. 3 is a schematic drawing depicting recombinant PAF-AH fragments andtheir catalytic activity;

FIG. 4 depicts mass spectroscopy results for a recombinant PAF-AHproduct, rPH.2.

FIG. 5 depicts mass spectroscopy results for a recombinant PAF-AHproduct, rPH.9.

FIG. 6 is a bar graph illustrating blockage of PAF-induced rat footedema by locally administered recombinant PAF-AH of the invention;

FIG. 7 is a bar graph illustrating blockage of PAF-induced rat footedema by intravenously administered PAF-AH;

FIG. 8 is a bar graph showing that PAF-AH blocks PAF-induced edema butnot zymosan A-induced edema;

FIGS. 9A and 9B present dose response results of PAF-AHanti-inflammatory activity in rat food edema;

FIGS. 10A and 10B present results indicating the in Vivo efficacy of asingle dose of PAF-AH over time:

FIG. 11 is a line graph representing the pharmacokinetics of PAF-AH inrat circulation; and

FIG. 12 is a bar graph showing the anti-inflammatory effects of PAF-AHin comparison to the lesser effects of PAF antagonists in rat footedema.

FIG. 13 presents results indicating that PAF-AH neutralizes theapoptotic effects of conditioned media from HIV-1-infected and activatedmonocytes.

DETAILED DESCRIPTION

The following examples illustrate the invention. Example 1 presents anovel method for the purification of PAF-AH from human plasma. Example 2describes amino acid microsequencing of the purified human plasmaPAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AHis described in Example 3. Identification of a putative splice variantof the human plasma PAF-AH gene is described in Example 4. The cloningof genomic sequences encoding human plasma PAF-AH is described inExample 5. Example 6 desribes the cloning of canine, murine, bovine,chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AHcDNA. Example 7 presents the results of an assay evidencing theenzymatic activity of recombinant PAF-AH transiently expressed in COS 7cells. Example 8 describes the expression of full length, truncated andchimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammaliancells. Example 9 presents protocols for purification of recombinantPAF-AH from E. coli and assays confirming its enzymatic activity.Example 10 describes various recombinant PAF-AH products including aminoacid substitution analogs and amino and carboxy-truncated products, anddescribes experiments demonstrating that native PAF-AH isolated fromplasma is glycosylated. Results of a Northern blot assay for expressionof human plasma PAF-AH RNA in various tissues and cell lines arepresented in Example 11 while results of in situ hybridization arepresented in Example 12. Example 13 describes the development ofmonoclonal and polyclonal antibodies specific for human plasma PAF-AH.Examples 14, 15, 16, 17, 18 and 19 respectively describe the in vivotherapeutic effect of administration of recombinant PAF-AH products ofthe invention on acute inflammation, pleurisy, asthma, necrotizingenterocolitis, adult respiratory distress syndrome and pancreatitis inanimal models. Example 20 describes the in vitro effect of recombinantPAF-AH product on neurotoxicity associated with HIV infection. Example21 presents the results of immunoassays of serum of human patientsexhibiting a deficiency in PAF-AH activity and describes theidentification of a genetic lesion in the patients which is apparentlyresponsible for the deficiency.

EXAMPLE 1

PAF-AH was purified from human plasma in order to provide material foramino acid sequencing.

A. Optimization of Purification Conditions

Initially, low density lipoprotein (LDL) particles were precipitatedfrom plasma with phosphotungstate and solubilized in 0.1% Tween 20 andsubjected to chromatography on a DEAE column (Pharmacia, Uppsala,Sweden) according to the method of Stafforini et al. (1987), supra, butinconsistent elution of PAF-AH activity from the DEAE column requiredreevaluation of the solubilization and subsequent purificationconditions.

Tween 20, CHAPS (Pierce Chemical Co., Rockford, Ill.) and octylglucoside were evaluated by centrifugation and gel filtrationchromatography for their ability to solubilize LDL particles. CHAPSprovided 25% greater recovery of solubilized activity than Tween 20 and300% greater recovery than octyl glucoside. LDL precipitate solubilizedwith 10 mM CHAPS was then fractionated on a DEAE Sepharose Fast Flowcolumn (an anion exchange column; Pharmacia) with buffer containing 1 mMCHAPS to provide a large pool of partially purified PAF-AH ("the DEAEpool") for evaluation of additional columns.

The DEAE pool was used as stating material to test a variety ofchromatography columns for utility in further purifying the PAF-AHactivity. The columns tested included: Blue Sepharose Fast Flow(Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow(Pharmacia), a cation exchange column; Cu Chelating Sepharose(Pharmacia), a metal ligand affinity column; Fractogel S (EMSeparations, Gibbstown, N.J.), a cation exchange column; andSephacryl-200 (Pharmacia), a gel filtration column. Thesechromatographic procedures all yielded low, unsatisfactory levels ofpurification when operated in 1 mM CHAPS. Subsequent gel filtrationchromatography on Sephacryl S-200 in 1 mM CHAPS generated anenzymatically active fraction which eluted over a broad size rangerather than the expected 44 kDa approximate size. Taken together, theseresults indicated that the LDL proteins were aggregating in solution.

Different LDL samples were therefore evaluated by analytical gelfiltration chromatography for aggregation of the PAF-AH activity.Samples from the DEAE pool and of freshly solubilized LDL precipitatewere analyzed on Superose 12 (Pharmacia) equilibrated in buffer with 1mM CHAPS. Both samples eluted over a very broad range of molecularweights with most of the activity eluting above 150 kDa. When thesamples were then analyzed on Superose 12 equilibrated with 10 mM CHAPS,the bulk of the activity eluted near 44 kDa as expected for PAF-AHactivity. However, the samples contained some PAF-AH activity in thehigh molecular weight region corresponding to aggregates.

Other samples eluted PAF-AH activity exclusively in the approximately 44kDa range when they were subsequently tested by gel filtration. Thesesamples were an LDL precipitate solubilized in 10 mM CHAPS in thepresence of 0.5M NaCl and a fresh DEAE pool that was adjusted to 10 mMCHAPS after elution from the DEAE column. These data indicate that atleast 10 mM CHAPS is required to maintain non-aggregated PAF-AH.Increase of the CHAPS concentration from 1 mM to 10 mM afterchromatography on DEAE but prior to subsequent chromatographic stepsresulted in dramatic differences in purification. For example, thedegree of PAF-AH purification on S-Sepharose Fast Flow was increasedfrom 2-fold to 10-fold. PAF-AH activity bound the Blue Sepharose FastFlow column irreversibly in 1 mM CHAPS, but the column provided thehighest level of purification in 10 mM CHAPS. The DEAE chromatographywas not improved with prior addition of 10 mM CHAPS.

Chromatography on Cu Chelating Sepharose after the Blue Sepharose FastFlow column concentrated PAF-AH activity 15-fold. It was also determinedthat PAF-AH activity could be recovered from a reducedSDS-polyacrylamide gel, as long as samples were not boiled. The activityof material eluted from the Cu Chelating Sepharose column when subjectedto SDS-polyacrylamide gel electrophoresis coincided with a major proteinband when the gel was silver stained.

B. PAF-AH Purification Protocol

The novel protocol utilized to purify PAF-AH for amino acid sequencingtherefore comprised the following steps which were performed at 4° C.Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottlesand adjusted to pH 8.6. LDL particles were then precipitated by adding90 ml of 3.85% sodium phosphotungstate followed by 23 ml of 2M MgCl₂.The plasma was then centrifuged for 15 minutes at 3600 g. Pellets wereresuspended in 800 ml of 0.2% sodium citrate. LDL was precipitated againby adding 10 g NaCl and 24 ml of 2M MgCl₂. LDL particles were pelletedby centrifugation for 15 minutes at 3600 g. This wash was repeatedtwice. Pellets were then frozen at -20° C. LDL particles from 5L ofplasma were resuspended in 5 L of buffer A (25 mM Tris-HCl, 10 mM CHAPS,pH 7.5) and stirred overnight. Solubilized LDL particles werecentrifuged at 3600 g for 1.5 hours. Supernatants were combined andfiltered with Whatman 113 filter paper to remove any remaining solids.Solubilized LDL supernatant was loaded on a DEAE Sepharose Fast Flowcolumn (11 cm×10 cm; 1 L resin volume; 80 ml/minute) equilibrated inbuffer B (25 mM Tris-HCl, 1 mM CHAPS, pH 7.5). The column was washedwith buffer B until absorbance returned to baseline. Protein was elutedwith an 8 L, 0-0.5M NaCl gradient and 480 ml fractions were collected.This step was necessary to obtain binding to the Blue Sepharose FastFlow column below. Fractions were assayed for acetylhydrolase activityessentially by the method described in Example 4.

Active fractions were pooled and sufficient CHAPS was added to make thepool about 10 mM CHAPS. The DEAE pool was loaded overnight at 4ml/minute onto a Blue Sepharose Past Flow column (5 cm×10 cm; 200 ml bedvolume) equilibrated in buffer A containing 0.5M NaCl. The column waswashed with the equilibration buffer at 16 ml/minute until absorbancereturned to baseline. PAF-AH activity was step eluted with buffer Acontaining 0.5M KSCN (a chaotropic salt) at 16 ml/minute and collectedin 50 ml fractions. This step resulted in greater than 1000-foldpurification. Active fractions were pooled, and the pool was adjusted topH 8.0 with 1M Tris-HCl pH 8.0. The active pool from Blue Sepharose FastFlow chromatography was loaded onto a Cu Chelating Sepharose column (2.5cm×2 cm; 10 ml bed volume; 4 ml/minute) equilibrated in buffer C [25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, pH 8.0 (pH 7.5 also worked)], and thecolumn was washed with 50 ml buffer C. PAF-AH activity was eluted with100 ml 50 mM imidazole in buffer C and collected in 10 ml fractions.Fractions containing PAF-AH activity were pooled and dialyzed againstbuffer A. In addition to providing a 15-fold concentration of PAF-AHactivity, the Cu Chelating Sepharose column gave a small purification.The Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15 minutesat 37° C. and loaded onto a 0.75 mm, 7.5% polyacrylamide gel. Gel sliceswere cut every 0.5 cm and placed in disposable microfuge tubescontaining 200 μl 25 mM Tris-HCl, 10 mM CHAPS, 150 mM NaCl. Slices wereground up and allowed to incubate overnight at 4° C. The supernatant ofeach gel slice was then assayed for PAF-AH activity to determine whichprotein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity wasfound in an approximately 44 kDa band. Protein from a duplicate gel waselectrotransferred to a PVDF membrane (Immobilon-P, Millipore) andstained with Coomassie Blue. A photograph of the PVDF membrane ispresented in FIG. 1.

As presented in Table 1 below, approximately 200 μg PAF-AH was purified2×10⁶ -fold from 5 L human plasma. In comparison, a 3×10⁴ -foldpurification of PAF-AH activity is described in Stafforini et al.(1987), supra.

                                      TABLE 1                                     __________________________________________________________________________                  Total                                                                             Prot.                                                                             Specific                                                         Activity                                                                           Activity                                                                          Conc.                                                                             Activity                                                                           % Recovery                                                                           Fold                                        Vol.     (cpm ×                                                                       (cpm ×                                                                      (mg/                                                                              (cpm ×                                                                       of Activity                                                                          Purification                                Sample                                                                              (ml)                                                                             10.sup.6)                                                                          10.sup.9)                                                                         ml) 10.sup.6)                                                                          Step                                                                             Cum.                                                                              Step                                                                              Cum.                                    __________________________________________________________________________    Plasma                                                                              5000                                                                             23   116 62  0.37 100                                                                              100 1    1                                      LDL   4500                                                                             22   97  1.76                                                                              12    84                                                                               84 33   33                                     DEAE  4200                                                                             49   207 1.08                                                                              46   212                                                                              178 3.7 124                                     Blue   165                                                                             881  14  0.02                                                                              54200                                                                               70                                                                              126 1190                                                                              1.5 × 10.sup.5                    Cu     12                                                                              12700                                                                              152 0.15                                                                              82200                                                                              104                                                                              131 1.5 2.2 × 10.sup.5                    SDS-PAGE                                                                            -- --   --  --  --   -- --  ˜10                                                                         2.2 × 10.sup.6                    __________________________________________________________________________

In summary, the following steps were unique and critical for successfulpurification of plasma PAF-AH for microsequencing: (1) solubilizationand chromotography in 10 mM CHAPS, (2) chromatography on a blue ligandaffinity column such as Blue Sepharose Fast Flow, (3) chromatography ona Cu ligand affinity column such as Cu Chelating Sepharose, and (4)elution of PAF-AH from SDS-PAGE.

EXAMPLE 2

For amino acid sequencing, the approximately 44 kDa protein band fromthe PAF-AH- containing PVDF membrane described in Example 1 was excisedand sequenced using an Applied Biosystems 473A Protein sequencer.N-terminal sequence analysis of the approximately 44 kDa protein bandcorresponding to the PAF-AH activity indicated that the band containedtwo major sequences and two minor sequences. The ratio of the two majorsequences was 1:1 and it was therefore difficult to interpret thesequence data.

To distinguish the sequences of the two major proteins which had beenresolved on the SDS gel, a duplicate PVDF membrane containing theapproximately 44 kDa band was cut in half such that the upper part andthe lower part of the membrane were separately subjected to sequencing.

The N-terminal sequence obtained for the lower half of the membrane was:

    F K D L G E E N F K A L V L I A F                                                                     SEQ ID NO: 1                                      

A search of protein databases revealed this sequence to be a fragment ofhuman serum albumin. The upper half of the same PVDF membrane was alsosequenced and the N-terminal amino acid sequence determined was:

    I Q V L M A A A S F G Q T K I P                                                                       SEQ ID NO: 2                                      

This sequence did not match any protein in the databases searched andwas different from the N-terminal amino acid sequence:

    M K P L V V F V L G G                                                                              SEQ ID NO: 3                                         

which was reported for erythrocyte cytoplasmic PAF-AH in Stafforini etal. (1993), supra. The novel sequence (SEQ ID NO: 2) was utilized forcDNA cloning of human plasma PAF-AH as described below in Example 3.

EXAMPLE 3

A full length clone encoding human plasma PAF-AH was isolated from amacrophage cDNA library.

A. Construction of a Macrophage cDNA Library

Poly A⁺ RNA was harvested from peripheral blood monocyte-derivedmacrophages. Double-stranded, blunt-ended cDNA was generated using theInvitrogen Copy Kit (San Diego, Calif.) and BstXI adapters were ligatedto the cDNA prior to insertion into the mammalian expression vector,pRc/CMV (Invitrogen). The resulting plasmids were introduced into E.coli strain XL-1 Blue by electroporation. Transformed bacteria wereplated at a density of approximately 3000 colonies per agarose plate ona total of 978 plates. Plasmid DNA prepared separately from each platewas retained in individual pools and was also combined into larger poolsrepresenting 300,000 clones each.

B. Library Screening by PCR

The macrophage library was screened by the polymerase chain reactionutilizing a degenerate antisense oligonucleotide PCR primer based on thenovel N-terminal amino acid sequence described in Example 2. Thesequence of the primer is set out below in IUPAC nomenclature and where"I" is an inosine.

    5' ACATGAATTCGGIATCYTTIGTYTGICCTAA 3'                                                                  SEQ ID NO: 4                                     

The codon choice tables of Wada et al., Nuc. Acids Res. 19S: 1981-1986(1991) were used to select nucleotides at the third position of eachcodon of the primer. The primer was used in combination with a primerspecific for either the SP6 or T7 promoter sequences, both of whichflank the cloning site of pRc/CMV, to screen the macrophage librarypools of 300,000 clones. All PCR reactions contained 100 ng of templatecDNA, 1 μg of each primer, 0.125 mM of each dNTP, 10 mM Tris-HCl pH 8.4,50 mM MgCl₂ and 2.5 units of Taq polymerase. An initial denaturationstep of 94° C. for four minutes was followed by 30 cycles ofamplification of 1 minute at 94° C., 1 minute at 60° C. and 2 minutes at72° C. The resulting PCR product was cloned into pBluescript SK⁻(Stratagene, La Jolla, Calif.) and its nucleotide sequence determined bythe dideoxy chain termination method. The PCR product contained thesequence predicted by the novel peptide sequence and corresponds tonucleotides 1 to 331 of SEQ ID NO: 7.

The PCR primers set out below, which are specific for the cloned PCRfragment described above, were then designed for identifying a fulllength clone.

    Sense Primer (SEQ ID NO: 5)                                                   5' TATTTCTAGAAGTGTGGTGGAACTCGCTGG 3'                                          Antisense Primer (SEQ ID NO: 6)                                               5' CGATGAATCAGCTTGCAGCAGCCATCAGTAC 3'                                     

PCR reactions utilizing the primers were performed as described above tofirst screen the cDNA pools of 300,000 clones and then the appropriatesubset of the smaller pools of 3000 clones. Three pools of 3000 cloneswhich produced a PCR product of the expected size were then used totransform bacteria.

C. Library Screening by Hybridization

DNA from the transformed bacteria was subsequently screened byhybridization using the original cloned PCR fragment as a probe.Colonies were blotted onto nitrocellulose and prehybridized andhybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodiumcitrate, 0.05M sodium phosphate pH 6.5, 1% polyvinyl pyrolidine, 1%Ficoll, 1% bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA.The hybridization probe was labeled by random hexamer priming. Afterovernight hybridization at 42° C., blots were washed extensively in0.03M sodium chloride, 3 mM sodium citrate, 0.1% SDS at 42° C. Thenucleotide sequence of 10 hybridizing clones was determined. One of theclones, clone sAH 406-3, contained the sequence predicted by theoriginal peptide sequence of the PAF-AH activity purified from humanplasma. The DNA and deduced amino acid sequences of the human plasmaPAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.

Clone sAH 406-3 contains a 1.52 kb insert with an open reading flamethat encodes a predicted protein of 441 amino acids. At the aminoterminus, a relatively hydrophobic segment of 41 residues precedes theN-terminal amino acid (the isoleucine at position 42 of SEQ ID NO: 8)identified by protein microsequencing. The encoded protein may thus haveeither a long signal sequence or a signal sequence plus an additionalpeptide that is cleaved to yield the mature functional enzyme. Thepresence of a signal sequence is one characteristic of secretedproteins. In addition, the protein encoded by clone sAH 406-3 includesthe consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that isbelieved to contain the active site serine of all known mammalianlipases, microbial lipases and serine proteases. See Chapus et al.,Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530(1988).

Table 2 below is a comparison of the amino acid composition of the humanplasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and theamino acid composition of the purportedly purified material described byStafforini et al. (1987), supra.

                  TABLE 2                                                         ______________________________________                                                   Clone sAH 406-3                                                                         Stafforini et al.                                        ______________________________________                                        Ala          26          24                                                   Asp & Asn    48          37                                                   Cys           5          14                                                   Glu & Gln    36          42                                                   Phe          22          12                                                   Gly          29          58                                                   His          13          24                                                   Ile          31          17                                                   Lys          26          50                                                   Leu          40          26                                                   Met          10           7                                                   Pro          15          11                                                   Arg          18          16                                                   Ser          27          36                                                   Thr          20          15                                                   Val          13          14                                                   Trp           7          Not determined                                       Tyr          14          13                                                   ______________________________________                                    

The amino acid composition of the mature form of the human plasma PAF-AHof the invention and the amino acid composition of the previouslypurified material that was purportedly the human plasma PAF-AH areclearly distinct.

When alignment of the Hattori et al., supra nucleotide and deduced aminoacid sequences of bovine brain cytoplasmic PAF-AH with the nucleotideand amino acid sequences of the human plasma PAF-AH of the invention wasattempted, no significant structural similarity in the sequences wasobserved.

EXAMPLE 4

A putative splice variant of the human PAF-AH gene was detected when PCRwas performed on macrophage and stimulated PBMC cDNA using primers thathybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ IDNO: 7) and the region spanning the translation termination codon at the3' end of the PAF-AH cDNA (nucleotides 1465 to 1487 of SEQ ID NO: 7).The PCR reactions yielded two bands on a gel, one corresponding to theexpected size of the PAF-AH cDNA of Example 3 and the other was about100 bp shorter. Sequencing of both bands revealed that the larger bandwas the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2(Example 5 below) of the PAF-AH sequence which encodes the putativesignal and pro-peptide sequences of plasma PAF-AH. The predictedcatalytic triad and all cysteines were present in the shorter clone,therefore the biochemical activity of the protein encoded by the cloneis likely to match that of the plasma enzyme.

To begin to assess the biological relevance of the PAF-AH splice variantthat is predicted to encode a cytoplasmically active enzyme, therelative abundance of the two forms in blood monocyte-derivedmacrophages was assayed by RNase protection. Neither message was presentin freshly isolated monocytes but both messages were found at day 2 ofin vitro differentiation of the monocytes into macrophages and persistedthrough 6 days of culture. The quantity of the two messages wasapproximately equivalent throughout the differentiation period. Incontrast, similar analyses of neural tissues revealed that only fulllength message predicted to encode the full length extracellular form ofPAF-AH is expressed.

EXAMPLE 5

Genomic human plasma PAF-AH sequences were also isolated. The structureof the PAF-AH gene was determined by isolating lambda and P1 phageclones containing human genomic DNA by DNA hybridization underconditions of high stringency. Fragments of the phage clones weresubcloned and sequenced using primers designed to anneal at regularintervals throughout the cDNA clone sAH 406-3. In addition, newsequencing primers designed to anneal to the intron regions flanking theexons were used to sequence back across the exon-intron boundaries toconfirm the sequences. Exon/intron boundaries were defined as the pointswhere the genomic and cDNA sequences diverged. These analyses revealedthat the human PAF-AH gene is comprised of 12 exons.

Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetalplacental library constructed in lamda FIX (Stratagene). Phage plaqueswere blotted onto nitrocellulose and prehybridized and hybridized in 50%formamide, 0.75M sodium chloride, 75 mM sodium citrate, 50 mM sodiumphosphate (pH 6.5), 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serumalbumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridizationprobe used to identify a phage clone containing exons 2-6 and part of 7consisted of the entire cDNA clone sAH 406-3. A clone containing exon 1was identified using a fragment derived from the 5' end of the cDNAclone (nucleotides 1 to 312 of SEQ ID NO: 7). Both probes were labelledwith ³² P by hexamer random priming. After overnight hybridization at42° C., blots were washed extensively in 30 mM sodium chloride, 3 mMsodium citrate, 0.1% SDS at 42° C. The DNA sequences of exons 1, 2, 3,4, 5, and 6 along with partial surrounding intron sequences are set outin SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.

The remainder of exon 7 as well as exons 8, 9, 10, 11, and 12 weresubcloned from a P1 clone isolated from a human P1 genomic library. P1phage plaques were blotted onto nitrocellulose and prehybridized andhybridized in 0.75M sodium chloride, 50 mM sodium phosphate (pH 7.4), 5mM EDTA, 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serum albumin,0.5% SDS, and 0.1 mg/ml total human DNA. The hybridization probe,labeled with ³² P by hexamer random priming, consisted of a 2.6 kb EcoR1fragment of genomic DNA derived from the 3' end of a lambda cloneisolated above. This fragment contained exon 6 and the part of exon 7present on the phage clone. After overnight hybridization at 65° C.,blots were washed as described above. The DNA sequences of exons 7, 8,9, 10, 11, and 12 along with partial surrounding intron sequences areset out in SEQ ID NOs: 15, 16, 17, 18, 19, and 20, respectively.

EXAMPLE 6

Full length plasma PAF-AH cDNA clones were isolated from mouse, canine,bovine and chicken spleen cDNA libraries and a partial rodent clone wasisolated from a rat thymus cDNA library. The clones were identified bylow stringency hybridization to the human cDNA (hybridization conditionswere the same as described for exons 1 through 6 in Example 5 aboveexcept that 20% formamide instead of 50% formamide was used). A 1 kbHindIII fragment of the human PAF-AH sAH 406-3 cDNA clone (nucleotides309 to 1322 of SEQ ID NO: 7) was used as a probe. In addition, a partialmonkey clone was isolated from macaque brain cDNA by PCR using primersbased on nucleotides 285 to 303 and 851 to 867 of SEQ ID NO: 7. Thenucleotide and deduced amino acid sequences of the mouse, canine,bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs:21, 22, 23, 24, 25, and 26, respectively.

A comparison of the deduced amino acid sequences of the cDNA clones withthe human cDNA clone results in the amino acid percentage identityvalues set out in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Human         Dog    Mouse     Bovine                                                                              Chicken                                  ______________________________________                                        Dog     80        100    64      82    50                                     Mouse   66        64     100     64    47                                     Monkey  92        82     69      80    52                                     Rat     74        69     82      69    55                                     Bovine  82        82     64      100   50                                     Chicken 50        50     47      50    100                                    ______________________________________                                    

About 38% of the residues are completely conserved in all the sequences.The most divergent regions are at the amino terminal end (containing thesignal sequence) and the carboxyl terminal end which are shown inExample 10 as not critical for enzymatic activity. TheGly-Xaa-Ser-Xaa-Gly motif (SEQ ID NO: 27) found in neutral lipases andother esterases was conserved in the bovine, canine, mouse, rat andchicken PAF-AH. The central serine of this motif serves as the activesite nucleophile for these enzymes. The predicted aspartate andhistidine components of the active site (Example 10A) were alsoconserved. The human plasma PAF-AH of the invention therefore appears toutilize a catalytic triad and may assume the α/β hydrolase conformationof the neutral lipases even though it does not exhibit other sequencehomology to the lipases.

Moreover, human plasma PAF-AH is expected to have a region that mediatesits specific interaction with the low density and high densitylipoprotein particles of plasma. Interaction with these particles may bemediated by the N-terminal half of the molecule which has largestretches of amino acids highly conserved among species but does notcontain the catalytic triad of the enzyme.

EXAMPLE 7

To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example3) encodes a protein having PAF-AH activity, the pRc/CMV expressionconstruct was transiently expressed in COS 7 cells. Three days followingtransfection by a DEAE Dextran method, COS cell media was assayed forPAF-AH activity.

Cells were seeded at a density of 300,000 cells per 60 mm tissue culturedish. The following day, the cells were incubated in DMEM containing 0.5mg/ml DEAE dextran, 0.1 mM chloroquine and 5-10 μg of plasmid DNA for 2hours. Cells were then treated with 10% DMSO in phosphate-bufferedsaline for 1 minute, washed with media and incubated in DMEM containing10% fetal calf serum previously treated with diisopropyl fluorophosphate(DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days ofincubation, media from transfected cells were assayed for PAF-AHactivity. Assays were conducted in the presence and absence of either 10mM EDTA or 1 mM DFP to determine whether the recombinant enzyme wascalcium-independent and inhibited by the serine esterase inhibitor DFPas previously described for plasma PAF-AH by Stafforini et al. (1987),supra. Negative controls included cells transfected with pRc/CMV eitherlacking an insert or having the sAH 406-3 insert in reverse orientation.

PAF-AH activity in transfectant supernatants was determined by themethod of Stafforini et al. (1990), supra, with the followingmodifications. Briefly, PAF-AH activity was determined by measuring thehydrolysis of ³ H-acetate from [acetyl-³ H] PAF (New England Nuclear,Boston, Mass.). The aqueous free ³ H-acetate was separated from labeledsubstrate by reversed-phase column chromatography over octadecylsilicagel cartridges (Baker Research Products, Phillipsburg, Pa.). Assays werecarried out using 10 μl transfectant supernatant in 0.1M Hepes buffer,pH 7.2, in a reaction volume of 50 μ. A total of 50 pmoles of substratewere used per reaction with a ratio of 1:5 labeled: cold PAF. Reactionswere incubated for 30 minutes at 37° C. and stopped by the addition of40 μl of 10M acetic acid. The solution was then washed through theoctadecylsilica gel cartridges which were then rinsed with 0.1M sodiumacetate. The aqueous eluate from each sample was collected and countedin a liquid scintillation counter for one minute. Enzyme activity wasexpressed in counts per minute.

As shown in FIG. 2, media from cells transfected with sAH 406-3contained PAF-AH activity at levels 4-fold greater than background. Thisactivity was unaffected by the presence of EDTA but was abolished by 1mM DFP. These observations demonstrate that clone sAH 406-3 encodes anactivity consistent with the human plasma enzyme PAF-AH.

EXAMPLE 8

Full length and various truncated human plasma PAF-AH DNAs and achimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast andstably expressed in mammalian cells by recombinant methods.

A. Expression in E. coli

PCR was used to generate a protein coding fragment of human plasmaPAF-AH cDNA from clone sAH 406-3 which was readily amenable tosubcloning into an E. coli expression vector. The subcloned segmentbegan at the 5' end of the human gene with the codon that encodes Ile₄₂(SEQ ID NO: 8), the N-terminal residue of the enzyme purified from humanplasma. The remainder of the gene through the native termination codonwas included in the construct. The 5' sense PCR primer utilized was:

    SEQ ID NO: 28                                                                 5'TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG                               

3' and contained an XbaI cloning site as well as a translationinitiation codon (underscored). The 3' antisense primer utilized was:

    SEQ ID NO: 29                                                                 5' ATTGATATCCTAATTGTATTTCTCTATTCCTG 3'                                    

and encompassed the termination codon of sAH 406-3 and contained anEcoRV cloning site. PCR reactions were performed essentially asdescribed in Example 3. The resulting PCR product was digested with XbaIand EcoRV and subcloned into a pBR322 vector containing the Trp promoter[deBoer et al., PNAS, 80:21-25 (1983)] immediately upstream of thecloning site. E. coli stain XL-1 Blue was transformed with theexpression construct, and cultured in L broth containing 100 μg/ml ofcarbenicillin. Transformants from overnight cultures were pelleted andresuspended in lysis buffer containing 50 mM Tris-HCl pH 7.5, 50 mMNaCl, 10 mM CHAPS, 1 mM EDTA, 100 μg/ml lysozyme, and 0.05trypsin-inhibiting units (TIU)/ml Aprotinin. Following a 1 hourincubation on ice and sonication for 2 minutes, the lysates were assayedfor PAF-AH activity by the method described in Example 4. E. colitransformed with the expression construct (designated trp AH) generateda product with PAF-AH activity. See Table 6 in Example 9.

Constructs including three additional promoters, the tacII promoter(deBoer, supra), the arabinose (ara) B promoter from Salmonellatyphimurium [Horwitz et al., Gene, 14: 309-319 (1981)], and thebacteriophage T7 promoter, were also utilized to drive expression ofhuman PAF-AH sequences in E. coli. Constructs comprising the Trppromoter (pUC trp AH), the tacII promoter (pUC tac AH), and the araBpromoter (pUC ara AH) were assembled in plasmid pUC19 (New EnglandBiolabs, Mass.) while the construct comprising the T7 promoter (pET AH)was assembled in plasmid pET15B (Novagen, Madison, Wis.). A constructcontaining a hybrid promoter, pHAB/PH, consisting of the araB promoterfused to the ribosome binding sites of the T7 promoter region was alsoassembled in pET15B. All E. coli constructs produced PAF-AH activitywithin a range of 20 to 50 U/ml/OD₆₀₀. This activity corresponded to atotal recombinant protein mass of ≧1% of the total cell protein.

Several. E. coli expression constructs were also evaluated which producePAF-AH with extended amino termini. The N-terminus of natural plasmaPAF-AH was identified as Ile₄₂ by amino acid sequencing (Example 2).However, the sequence immediately upstream of Ile₄₂ does not conform toamino acids found at signal sequence cleavage sites [i.e., the"-3-1-rule" is not followed, as lysine is not found at position -1; seevon Heijne, Nuc. Acids Res., 14:4683-4690 (1986)]. Presumably a moreclassical signal sequence (M₁ -A₁₇ or M₁ -P₂₁) is recognized by thecellular secretion system, followed by endoproteolytic cleavage. Theentire coding sequence for PAF-AH beginning at the initiating methionine(nucleotides 162 to 1487 of SEQ ID NO: 7) was engineered for expressionin E. coli using the trp promoter. As shown in Table 4, this constructmade active PAF-AH, but expression was at about one fiftieth of thelevel of the original construct beginning at Ile₄₂. Another expressionconstruct, beginning at Val₁₈ (nucleotides 213 to 1487 of SEQ ID NO: 7),produced active PAF-AH at about one third the level of the originalconstruct. These results suggest that amino terminal end extensions arenot critical or necessary for activity of recombinant PAF-AH produced inE. coli.

                  TABLE 4                                                         ______________________________________                                                           PAF-AH activity                                                               (U/ml/OD.sub.600)                                          Construct            Lysate  Media                                            ______________________________________                                        pUC trp AH (Ile.sub.42 N-terminus)                                                                 177.7   0.030                                            pUC trp AH Met.sub.1 3.1     0.003                                            pUC trp AH Val.sub.18                                                                              54.6    0.033                                            ______________________________________                                    

Truncated recombinant human PAF-AH products were also produced in E.coli using a low copy number plasmid and a promoter that can be inducedby the addition of arabinose to the culture. One such N-terminallytruncated PAF-AH product is the recombinant expression product of DNAencoding amino acid residues Met₄₆ through Asn₄₄₁ of the polypeptideencoded by full length PAF-AH cDNA (SEQ ID NO: 8), and is designatedrPH.2. The plasmid used for production of rPH.2 in bacterial cells waspBAR2/PH.2, a pBR322-based plasmid that carries (1) nucleotides 297 to1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with the methioninecodon at position 46, (2) the araB-C promoters and araC gene from thearabinose operon of Salmonella typhimurium, (3) a transcriptiontermination sequence from the bacteriophage T7, and (4) a replicationorigin from bacteriophage f1.

Specifically, pBAR2/PH.2 included the following segments of DNA: (1)from the destroyed AatII site at position 1994 to the EcoRI site atnucleotide 6274, vector sequence containing an origin of replication andgenes encoding resistance to either ampicillin or tetracycline derivedfrom the bacterial plasmid pBR322; (2) from the EcoRI site at position6274 to the XbaI site at position 131, DNA from the Salmonellatyphimurium arabinose operon (Genbank accession numbers M11045, M11046,M11047, J01797); (3) from the XbaI site at position 131 to the NcoI siteat position 170, DNA containing a ribosome binding site from pET-21b(Novagen, Madison, Wis.); (4) from the NcoI site at position 170 to theXhoI site at position 1363, human PAF-AH cDNA sequence; and (5) from theXhoI site at position 1363 to the destroyed AatII site at position 1993,a DNA fragment from pET-21b (Novagen) that contains a transcriptiontermination sequence from bacteriophae T7 and an origin of replicationfrom bacteriophage f1.

Another PAF-AH product, designated rPH.9, is the recombinant expressionproduct of DNA encoding amino acid residues Met₄₆ through Ile₄₂₉ of thepolypeptide encoded by full length PAF-AH cDNA (SEQ ID NO: 8). The DNAencoding rPH.9 was inserted into the same vector used for production ofrPH.2 in bacterial cells. This plasmid was designated pBAR2/PH.9 andspecifically included the following segments of DNA: (1) from thedestroyed AatII site at position 1958 to the EcoRI site at nucleotide6239 of the vector sequence containing an origin of replication andgenes encoding resistance to either ampicillin or tetracycline derivedfrom the bacterial plasmid pBR322; (2) from the EcoRI site at position6239 to the XbaI site at position 131, DNA from the Salmonellatyphimurium arabinose operon (Genbank accession numbers M11045, M11046,M11047, J01797); (3) from the XbaI site at position 131 to the NcoI siteat position 170, DNA containing a ribosome binding site from pET-21b(Novagen, Madison, Wis.); (4) from the NcoI site at position 170 to theXhoI site at position 1328, human PAF-AH DNA sequence; (5) from the XhoIsite at position 1328 to the destroyed AatII site at position 1958, aDNA fragment from pET-21b (Novagen, Madison, Wis.) that contains atranscription termination sequence from bacteriophage T7 and a origin ofreplication from bacteriophage f1.

Expression of PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is under thecontrol of the araB promoter, which is tightly repressed in the presenceof glucose and absence of arabinose, but functions as a strong promoterwhen L-arabinose is added to cultures depleted of glucose. Selection forcells containing the plasmid can be accomplished through the addition ofeither ampicillin (or related antibiotics) or tetracycline to theculture medium. A variety of E. coli strains can be used as a host forrecombinant expression of PAF-AH products, including but not limited tostrains prototrophic for arabinose metabolism such as W3110, DH5α, BL21,C600, JM101 and their derivatives, strains containing mutations reducingproteolysis such as CAG629, KY1429, and strains defective in theirability to degrade arabinose such as SB7219 and MC1061. The advantage ofusing a strain that is unable to break down arabinose is that theinducer (arabinose) for production of PAF-AH is not depleted from themedium during the induction period, resulting in higher levels of PAF-AHcompared to that obtained with strains that are capable of metabolizingarabinose. Any suitable media and culturing conditions may be used toexpress active PAF-AH products in various E. coli strains. For example,either rich media formulations such LB, EDM295 (a M9 based minimummedium supplemented with yeast extract and acid hydrolysed casein), or"defined" media such as A675, an A based minimal medium set at pH 6.75employing glycerol as a carbon source and supplemented with traceelements and vitamins, permit substantial production of rPAF-AHproducts. Tetracycline is included in the media to maintain selection ofthe plasmid.

The plasmid pBAR2/PH.2 was transformed into the E. coli strain MC1061(ATCC 53338), which carries a deletion of the arabinose operon andthereby cannot metabolize arabinose. MC1061 is also a leucine auxotrophand was cultivated by batch-fed process using a defined media containingcasamino acids that complement the leucine mutation.

The E. coli M1061 cells transformed with pBAR2/PH.2 were grown at 30° C.in batch media containing 2 gm/L glucose. Glucose serves the dualpurpose of carbon source for cell growth, and repressor of the arabinosepromoter. When batch glucose levels were depleted (<50 mg/L), a nutrientfeed (containing 300 gm/L glucose) was started. The feed was increasedlinearly for 16 hours at a rate which limited acid bi-product formation.At this point, the nutrient feed was switched to media containingglycerol instead of glucose. Simultaneously, 500 gm/L L-arabinose wasadded to a final concentration of 5 gm/L. The glycerol feed was kept ata constant feed rate for 22 hours. Cells were harvested usinghollow-fiber filtration to concentrate the suspension approximately10-fold. Cell paste was stored at -70° C. A final cell mass of about 80gm/L was obtained (OD₆₀₀ =50-60) with a PAF-AH activity of 65-70 U/OD/mlrepresenting about 10% of total cell protein. The final culture volumeof about 75 liters contained 50-60 gm PAF-AH.

High level production of rPAF-AH products can be achieved whenpBAR2/PH.2 or PH.9 is expressed by strains SB7219 or MC1061. Otherstrains deficient in arabinose degradation are suitable for high celldensity production. Preferably, the cells are cultured under thefollowing conditions. Exponentially growing SB7219;pBAR2/PH.2 andSB7219;pBAR2/PH.9 strains are seeded into fermentors containing batchmedium containing 2 g/L glucose. Once glucose is consumed, the tanks arefed with a glycerol solution containing trace elements, vitamins,magnesium and ammonium salt to maintain healthy exponential growth. Thetanks are maintained at 30° C., provided air to supply oxygen andagitated to maintain the dissolved oxygen level above about 15%saturation. When the cell density of the culture is above 110 g/L (wetcell mass), constant feed rate is imposed and a bolus addition ofL-arabinose is added to the culture (about 0.5% final). Productformation is observed for 16-22 hours. The cultures typically achieve40-50 g/L (dry cell weight). Cells are harvested by centrifugation,stored at -70° C., and rPAF-AH product purified for analysis. Specificproductivities in excess of 150 units/ml/OD₆₀₀ are routinely obtained.

B. Expression in Yeast Cells

Recombinant human PAF-AH was also expressed in Saccharomyces cerevisiae.The yeast ADH2 promoter was used to drive rPAF-AH expression andproduced 7 U/ml/OD₆₀₀ (Table 5 below).

                  TABLE 5                                                         ______________________________________                                                                          Enzyme                                                                        Activity                                    Construct  Promoter  Strain       (U/ml/OD)                                   ______________________________________                                        pUC tac AH tac       E. coli W3110                                                                              30                                          pUC trp AH trp       E. coli W3110                                                                              40                                          pUC ara AH araB      E. coli W3110                                                                              20                                          pET AH     T7        E. coli BL21 (DE3)                                                                         50                                                               (Novagen)                                                pHAB/PH    araB/T7   E. coli XL-1 34                                          pBAR2/PH.2 araB      MC1061       90                                          pYep ADH2 AH                                                                             ADH2      Yeast BJ2.28  7                                          ______________________________________                                    

C. Expression of PAF-AH in mammalian cells

1. Expression of Human PAF-AH cDNA Constructs

Plasmids constructed for expression of PAF-AH, with the exception ofpSFN/PAFAH.1, employ a strong viral promoter from cytomegalovirus, apolyadenylation site from the bovine growth hormone gene, and the SV40origin of replication to permit high copy number replication of theplasmid in COS cells. Plasmids were electroporated into cells.

A first set of plasmids was constructed in which the 5' flankingsequence (pDC1/PAFAH.1) or both the 5' or 3' flanking sequences(PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flankingsequences from other genes known to be expressed at high levels inmammalian cells. Transfection of these plasmids into COS, CHO or 293cells led to production of PAF-AH at about the same level (0.01 units/mlor 24 fold above background) as that cited for clone sAH 406-3 inExample 7 after transient transfection of COS cells. Another plasmid wasconstructed which included a Friend spleen focus-forming virus promoterinstead of the cytomegalovirus promoter. The human PAF-AH cDNA wasinserted into plasmid pmH-neo [Hahn et al., Gene, 127: 267 (1993)] undercontrol of the Friend spleen focus-forming virus promoter. Transfectionof the myeloma cell line NS0 with the plasmid which was designatedpSFN/PAFAH.1 and screening of several hundred clones resulted in theisolation of two transfectants (4B11 and 1C11) that made 0.15-0.5units/ml of PAF-AH activity. Assuming a specific activity of 5000units/milligram, the productivity of these two NS0 transfectantscorresponds to about 0.1 mg/liter.

2. Expression of Mouse-Human Chimeric PAF-AH Gene Constructs

A construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in themammalian expression vector pRc/CMV resulted in production of secretedPAF-AH at the level of 5-10 units/ml (1000 fold above background) aftertransfection into COS cells. Assuming that the specific activity of themouse PAF-AH is about the same as that of the human enzyme, the mousecDNA is therefore expressed at a 500-1000 fold higher level than is thehuman PAF-AH cDNA.

To examine the difference between the expression levels of human andmouse PAF-AH in COS cells, two mouse-human chimeric genes wereconstructed and tested for expression in COS cells. The first of theseconstructs, pRc/PH.MHC1, contains the coding sequence for the N-terminal97 amino acids of the mouse PAF-AH polypeptide (SEQ ID NO: 21) fused tothe C-terminal 343 amino acids of human PAF-AH in the expression vectorpRc/CMV (Invitrogen, San Diego, Calif.). The second chimeric gene, inplasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40amino acids of the mouse PAF-AH polypeptide fused to the C-terminal 400residues of human PAF-AH in pRc/CMV. Transfection of COS cells withpRc/PH.MHC1 led to accumulation of 1-2 units/ml of PAF-AH activity inthe media. Conditioned media derived from cells transfected withpRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity.From these experiments, it appears that the difference in expressionlevel between mouse and human PAF-AH genes is attributable at least inpart to the polypeptide segment between the residues 40 and 97, or thecorresponding RNA or DNA segment encoding this region of the PAF-AHprotein.

3. Recoding of the First 290 bp of the PAF-AH Coding Sequence

One hypothesis for the low level of human PAF-AH synthesized intransfected mammalian cells is that the codons utilized by the naturalgene are suboptimal for efficient expression. However, it does not seemlikely that codon usage can account for 500-1000 fold difference inexpression levels between the mouse and human genes because optimizingcodons generally has at most only a 10-fold effect on expression. Asecond hypothesis to explain the difference between the mouse and humanPAF-AH expression levels is that the human PAF-AH mRNA in the 5' codingregion forms a secondary structure that leads to either relatively rapiddegradation of the mRNA or causes inefficient translation initiation orelongation.

To test these hypotheses, a synthetic fragment encoding the authentichuman PAF-AH protein from the amino-terminus to residue 96 but in whichmost of the codons have been substituted ("recoded") with a codon of adifferent sequence but encoding the same amino acid was constructed.Changing the second codon from GTG to GTA resulted in the creation of anAsp718 site, which was at one end of the synthetic fragment and which ispresent in the mouse cDNA. The other end of the fragment contained theBamHI site normally found at codon 97 of the human gene. Theapproximately 290 bp Asp718/BamHI fragment was derived from a PCRfragment that was made using the dual asymmetric PCR approach forconstruction of synthetic genes described in Sandhu et al.,Biotechniques, 12: 14-16 (1992). The synthetic Asp718/BamHI fragment wasligated with DNA fragments encoding the remainder of the human PAF-AHmolecule beginning with nucleotide 453 of SEQ ID NO: 7 such that asequence encoding authentic human PAF-AH enzyme was inserted into themammalian expression vector pRc/CMV (Invitrogen, San Diego) to createplasmid pRc/HPH.4. The complete sequence of the recoded gene is set outin SEQ ID NO: 30. The 5' flanking sequence adjacent to the human PAF-AHcoding sequence in pRc/HPH.4 is from that of a mouse cDNA encodingPAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).

To test expression of human PAF-AH from pRc/HPH.4, COS cells weretransiently transfected with pRc/HPH.4 (recoded human gene), pRc/MS9(mouse PAF-AH), or pRc/PH.MHC1 (mouse-human hybrid 1). The conditionedmedia from the transfected cells were tested for PAF-AH activity andfound to contain 5.7 units/ml (mouse gene), 0.9 units/ml (mouse-humanhybrid 1), or 2.6 units/ml (recoded human gene). Thus, the strategy ofrecoding the first 290 bp of coding sequence of human PAF-AH wassuccessful in boosting expression levels of human PAF-AH from a fewnanograms/ml to about 0.5 microgram/ml in a transient COS celltransfection. The recoded PAF-AH gene from pRc/HPH.4 will be insertedinto a mammalian expression vector containing the dihydrofolatereductase (DHFR) gene and DHFR-negative chinese hamster ovary cells willbe transfected with the vector. The transfected cells will be subjectedto methotrexate selection to obtain clones making high levels of humanPAF-AH due to gene amplification.

EXAMPLE 9

Recombinant human plasma PAF-AH (beginning at Ile₄₂) expressed in E.coli was purified to a single Coomassie-stained SDS-PAGE band by variousmethods and assayed for activities exhibited by the native PAF-AHenzyme.

A. Purification of Recombinant PAF-AH

The first purification procedure utilized is similar to that describedin Example 1 for native PAF-AH. The following steps were performed at 4°C. Pellets from 50 ml PAF/AH producing E. coli (transformed withexpression construct trp AH) were lysed as described in Example 8.Solids were removed by centrifugation at 10,000 g for 20 minutes. Thesupernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flowcolumn (2.5 cm×4 cm; 20 ml bed volume) equilibrated in buffer D (25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, pH 7.5). The column was washed with100 ml buffer D and eluted with 100 ml buffer A containing 0.5M KSCN at3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml CuChelating Sepharose column equilibrated in buffer D. The column waswashed with 5 ml buffer D followed by elution with 5 ml of buffer Dcontaining 100 mM imidazole with gravity flow. Fractions containingPAF-AH activity were analyzed by SDS-PAGE.

The results of the purification are shown in Table 6 wherein a unitequals μmol PAF hydrolysis per hour. The purification product obtainedat 4° C. appeared on SDS-PAGE as a single intense band below the 43 kDamarker with some diffuse staining directly above and below it. Therecombinant material is significantly more pure and exhibits greaterspecific activity when compared with PAF-AH preparations from plasma asdescribed in Example 1.

                                      TABLE 6                                     __________________________________________________________________________                  Total                                                                             Prot.                                                                     Act.                                                                              Conc.                                                                             Specific                                                                           % Recovery                                                                           Fold                                        Vol.     Activity                                                                           (units ×                                                                    (mg/                                                                              Activity                                                                           of Activity                                                                          Purification                                Sample                                                                              (ml)                                                                             (units/ml)                                                                         10.sup.3)                                                                         mL) (units/mg)                                                                         Step                                                                             Cum.                                                                              Step                                                                              Cum.                                    __________________________________________________________________________    Lysate                                                                              4.5                                                                               989 4451                                                                              15.6                                                                               63  100                                                                              100 1   1                                       Blue  15  64   960                                                                              0.07                                                                               914  22                                                                              22  14.4                                                                              14.4                                    Cu    1  2128 2128                                                                              0.55                                                                              3869 220                                                                              48  4.2 61                                      __________________________________________________________________________

When the same purification protocol was performed at ambienttemperature, in addition to the band below the 43 kDa marker, a group ofbands below the 29 kDa marker correlated with PAF-AH activity of assayedgel slices. These lower molecular weight bands may be proteolyticfragments of PAF-AH that retain enzymatic activity.

A different purification procedure was also performed at ambienttemperature. Pellets (100 g) of PAF-AH-producing E. coli (transformedwith the expression construct pUC trp AH) were resuspended in 200 ml oflysis buffer (25 mM Tris, 20 mM CHAPS, 50 mM NaCl, 1 mM EDTA, 50 μg/mlbenzamidine, pH 7.5) and lysed by passing three times through amicrofluidizer at 15,000 psi. Solids were removed by centrifugation at14,300×g for 1 hour. The supernatant was diluted 10-fold in dilutionbuffer [25 mM MES (2-[N-morpholino] ethanesulfonic acid), 10 mM CHAPS, 1mM EDTA, pH 4.9] and loaded at 25 ml/minute onto an S Sepharose FastFlow Column (200 ml) (a cation exchange column) equilibrated in Buffer E(25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH 5.5). The column waswashed with 1 liter of Buffer E, eluted with 1M NaCl, and the eluate wascollected in 50 ml fractions adjusted to pH 7.5 with 0.5 ml of 2M Trisbase. Fractions containing PAF-AH activity were pooled and adjusted to0.5M NaCl. The S pool was loaded at 1 ml/minute onto a Blue SepharoseFast Flow column (2.5 cm×4 cm; 20 ml) equilibrated in Buffer F (25 mMTris, 10 mM CHAPS, 0.5M NaCl, 1 mM EDTA, pH 7.5). The column was washedwith 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaClat 4 ml/minute. The Blue Sepharose Fast Flow chromatography step wasthen repeated to reduce endotoxin levels in the sample. Fractionscontaining PAF-AH activity were pooled and dialyzed against Buffer G (25mM Tris pH 7.5, 0.5M NaCl, 0.1% Tween 80, 1 mM EDTA).

The results of the purification are shown in Table 7 wherein a unitequals μmol PAF hydrolysis per hour.

                                      TABLE 7                                     __________________________________________________________________________                  Total                                                                             Prot                                                        Vol-          Act.                                                                              Conc                                                                              Specific                                                                           % Recovery                                                                           Fold                                        ume      Activity                                                                           (units ×                                                                    (mg/                                                                              Activity                                                                           of Activity                                                                          Purification                                Sample                                                                              (ml)                                                                             (units/ml)                                                                         10.sup.3)                                                                         mL) (units/mg)                                                                         Step                                                                             Cum.                                                                              Step                                                                              Cum.                                    __________________________________________________________________________    Lysate                                                                              200                                                                              5640 1128                                                                              57.46                                                                              98  100                                                                              100 1    1                                      S     111                                                                              5742  637                                                                              3.69                                                                              1557 57 56  16  16                                      Blue  100                                                                              3944  394                                                                              0.84                                                                              4676 35 62  3   48                                      __________________________________________________________________________

The purification product obtained appeared on SDS-PAGE as a singleintense band below the 43 kDa marker with some diffuse staining directlyabove and below it. The recombinant material is significantly more pureand exhibits greater specific activity when compared with PAF-AHpreparations from plasma as described in Example 1.

Yet another purification procedure contemplated by the present inventioninvolves the following cell lysis, clarification, and first columnsteps. Cells are diluted 1:1 in lysis buffer (25 mM Tris pH 7.5, 150 mMNaCl, 1% Tween 80, 2 mM EDTA). Lysis is performed in a chilledmicrofluidizer at 15,000-20,000 psi with three passes of the material toyield >99% cell breakage. The lysate is diluted 1:20 in dilution buffer(25 mM Tris pH 8.5, 1 mM EDTA) and applied to a column packed withQ-Sepharose Big Bead chromatography media (Pharmacia) and equilibratedin 25 mM Tris pH 8.5, 1 mM EDTA, 0.015% Tween 80. The eluate is diluted1:10 in 25 mM MES pH 5.5, 1.2M Ammonium sulfate, 1 mM EDTA and appliedto Butyl Sepharose chromography media (Pharmacia) equilibrated in thesame buffer. PAF-AH activity is eluted in 25 mM MES pH 5.5, 0.1% Tween80, 1 mM EDTA.

Still another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli includes the steps of: (a)preparing an E. coli extract which yields solubilized PAF-AH supernatantafter lysis in a buffer containing CHAPS; (b) dilution of the saidsupernatant and application to a anion exchange column equilibrated atabout pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column;(d) applying said adjusted eluate from said anion exchange column to ablue dye ligand affinity column; (e) eluting the said blue dye ligandaffinity column using a buffer comprising 3.0M salt; (f) dilution of theblue dye eluate into a suitable buffer for performing hydroxylapatitechromatography; (g) performing hydroxylapatite chromatography wherewashing and elution is accomplished using buffers (with or withoutCHAPS); (h) diluting said hydroxylapatite eluate to an appropriate saltconcentration for cation exchange chromatography; (i) applying saiddiluted hydroxylapatite eluate to a cation exchange column at a pHranging between approximately 6.0 to 7.0; (j) elution of PAF-AH fromsaid cation exchange column with a suitable formulation buffer, (k)performing cation exchange chromatography in the cold; and (l)formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step (a) above the lysis buffer is 25 mM Tris, 100 mMNaCl, 1 mM EDTA, 20 mM CHAPS, pH 8.0; in step (b) the dilution of thesupernatant for anion exchange chromatography is 3-4 fold into 25 mMTris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 and the column is a Q-Sepharosecolumn equilibrated with 25 mM Tris, 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS,pH 8.0; in step (c) the anion exchange column is eluted using 25 mMTris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0; in step (d) theeluate from step (c) is applied directly onto a blue dye affinitycolumn; in step (e) the column is eluted with 3M NaCl, 10 mM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate forhydroxylapatite chromatography is accomplished by dilution into 10 mMsodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH 6.2; in step (g)hydroxylapatite chromatography is accomplished using a hydroxylapatitecolumn equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mMCHAPS and elution is accomplished using 50 mM sodium phosphate, 100 mMNaCl (with or without) 10 mM CHAPS, pH 7.5; in step (h) dilution of saidhydroxylapatite eluate for cation exchange chromatography isaccomplished by dilution into a buffer ranging in pH from approximately6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step(i) a S Sepharose column is equilibrated with 50 mM sodium phosphate,(with or without) 10 mM CHAPS, pH 6.8; in step (j) elution isaccomplished with a suitable formulation buffer such as potassiumphosphate 50 mM, 12.5 mM aspartic acid, 125 mM NaCl, pH 7.5 containing0.01% Tween-80; and in step (k) cation exchange chromatrography isaccomplished at 2-8° C. Examples of suitable formulation buffers for usein step (l) which stabilize PAF-AH include 50 mM potassium phosphate,12.5 mM Aspartic acid, 125 mM NaCl pH 7.4 (approximately, with andwithout the addition of Tween-80 and or Pluronic F68) or 25 mM potassiumphosphate buffer containing (at least) 125 mM NaCl, 25 mM arginine and0.01% Tween-80 (with or without Pluronic F68 at approximately 0.1 and0.5%).

B. Activity of Recombinant PAF-AH

The most remarkable property of the PAF acetylhydrolase is its markedspecificity for substrates with a short residue at the sn-2 position ofthe substrate. This strict specificity distinguishes PAF acetylhydrolasefrom other forms of PLA₂. Thus, to determine if recombinant PAF-AHdegrades phospholipids with long-chain fatty acids at the sn-2 position,hydrolysis of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine(arachidonoylPC) was assayed since this is the preferred substrate for awell-characterized form of PLA₂. As predicted from previous studies withnative PAF-AH, this phospholipid was not hydrolyzed when incubated withrecombinant PAF-AH. In additional experiments, arachidonoylPC wasincluded in a standard PAF hydrolysis assay at concentrations rangingfrom 0 to 125 μM to determine whether it inhibited the hydrolysis of PAFby recombinant PAF-AH. There was no inhibition of PAF hydrolysis even atthe highest concentration of PAF-AH, which was 5-fold greater than theconcentration of PAF. Thus, recombinant PAF-AH exhibits the samesubstrate selectivity as the native enzyme; long chain substrates arenot recognized. Moreover, recombinant PAF-AH enzyme rapidly degraded anoxidized phospholipid (glutaroylPC) which had undergone oxidativecleavage of the sn-2 fatty acid. Native plasma PAF-AH has several otherproperties that distinguish it from other phospholipases includingcalcium-independence and resistance to compounds that modify sulfhydrylgroups or disrupt disulfides.

Both the native and recombinant plasma PAF-AH enzymes are sensitive toDFP, indicating that a serine comprises part of their active sites. Anunusual feature of the native plasma PAF acetylhydrolase is that it istightly associated with lipoproteins in circulation, and its catalyticefficiency is influenced by the lipoprotein environment. Whenrecombinant PAF-AH of the invention was incubated with human plasma(previously treated with DFP to abolish the endogenous enzyme activity),it associated with low and high density lipoproteins in the same manneras the native activity. This result is significant because there issubstantial evidence that modification of low density lipoproteins isessential for the cholesterol deposition observed in atheromas, and thatoxidation of lipids is an initiating factor in this process. PAF-AHprotects low density lipoproteins from modification under oxidizingconditions in vitro and may have such a role in vivo. Administration ofPAF-AH is thus indicated for the suppression of the oxidation oflipoproteins in atherosclerotic plaques as well as to resolveinflammation.

These results all confirm that the cDNA clone sAH 406-3 encodes aprotein with the activities of the the human plasma PAF acetylhydrolase.

EXAMPLE 10

Various other recombinant PAF-AH products were expressed in E. coli. Theproducts included PAF-AH analogs having single amino acid mutations andPAF-AH fragments.

A. PAF-AH Amino Acid Substitution Products

PAF-AH is a lipase because it hydrolyses the phospholipid PAF. While noobvious overall similarity exists between PAF-AH and other characterizedlipases, there are conserved residues found in comparisons ofstructurally characterized lipases. A serine has been identified as amember of the active site. The serine, along with an aspartate residueand a histidine residue, form a catalytic triad which represents theactive site of the lipase. The three residues are not adjacent in theprimary protein sequence, but structural studies have demonstrated thatthe three residues are adjacent in three dimensional space. Comparisonsof structures of mammalian lipases suggest that the aspartate residue isgenerally twenty-four amino acids C-terminal to the active site serine.In addition, the histidine is generally 109 to 111 amino acidsC-terminal to the active site serine.

By site-directed mutagenesis and PCR, individual codons of the humanPAF-AH coding sequence were modified to encode alanine residues and wereexpressed in E. coli. As shown in Table 8 below wherein, for example,the abbreviation "S108A" indicates that the serine residue at position108 was changed to an alanine, point mutations of Ser₂₇₃, Asp296, orHis₃₅₁ completely destroy PAF-AH activity. The distances between activesite residues is similar for PAF-AH (Ser to Asp, 23 amino acids; Ser toHis, 78 amino acids) and other lipases. These experiments demonstratethat Ser₂₇₃, Asp₂₉₆, and His₃₅₁ are critical residues for activity andare therefore likely candidates for catalytic triad residues. Cysteinesare often critical for the functional integrity of proteins because oftheir capacity to form disulfide bonds. The plasma PAF-AH enzymecontains five cysteines. To determine whether any of the five iscritical for enzyme actvity, each cysteine was mutated individually to aserine and the resulting mutants were expressed in E. coli. Preliminaryactivity results using partially purified preparations of theserecombinantly produced mutants are shown below in the second column ofTable 8, while results using more purified preparations are shown belowin the third column of Table 8. The data show that all of the cysteinemutants had largely equivalent activity, so that none of the cysteinesappears to be necessary for PAF-AH activity. Other point mutations alsohad little or no effect on PAF-AH catalytic activity. In Table 8, "++++"represents wild type PAF-AH activity of about 40-60 U/ml/OD₆₀₀, "+++"represents about 20-40 U/ml/OD₆₀₀ activity, "++" represents about 10-20U/ml/OD₆₀₀ activity, "+" represents 1-10 U/ml/OD₆₀₀ activity, and "-"indicates <1 U/ml/OD₆₀₀ activity.

                  TABLE 8                                                         ______________________________________                                                                 Specific PAF-AH                                                      PAF-AH   activity of                                          Mutation        activity purified preparations                                ______________________________________                                        Wildtype        ++++     6.9 mmol/mg/hr                                       S108A           ++++                                                          S273A           -                                                             D286A           -                                                             D286N           ++                                                            D296A           -                                                             D304A           ++++                                                          D338A           ++++                                                          H351A           -                                                             H395A, H399A    ++++                                                          C67S            +++      5.7 mmol/mg/hr                                       C229S           +        6.5 mmol/mg/hr                                       C291S           +        5.9 mmol/mg/hr                                       C334S           ++++     6.8 mmol/mg/hr                                       C407S           +++      6.4 mmol/mg/hr                                       C67S, C334S, C407S       6.8 mmol/mg/hr                                       ______________________________________                                    

B. PAF-AH Fragment Products

C-terminal deletions were prepared by digesting the 3' end of the PAF-AHcoding sequence with exonuclease III for various amounts of time andthen ligating the shortened coding sequence to plasmid DNA encoding stopcodons in all three reading frames. Ten different deletion constructswere characterized by DNA sequence analysis, protein expression, andPAF-AH activity. Removal of twenty-one to thirty C-terminal amino acidsgreatly reduced catalytic activity and removal of fifty-two residuescompletely destroyed activity. See FIG. 3.

Similar deletions were made at the amino terminal end of PAF-AH. Fusionsof PAF-AH with E. coli thioredoxin at the N-terminus were prepared tofacilitate consistent high level expression PAF-AH activity [LaVallie etal., Bio/technology, 11:187-193 (1993)]. Removal of nineteen amino acidsfrom the naturally processed N-terminus (Ile₄₂) reduced activity by 99%while removal of twenty-six amino acids completely destroyed enzymaticactivity in the fusion protein. See FIG. 3. Deletion of twelve aminoacids appeared to enhance enzyme activity about four fold.

In subsequent purifications of PAF-AH from fresh human plasma by amethod similar to that described in Example 1 Microcon 30 filter fromAmicon were utilized to concentrate Blue sepharose eluate instead of aCu column), two N-termini in addition to Ile₄₂ were identified, Ser₃₅and Lys₅₅. The heterogeneity may be the natural state of the enzyme inplasma or may occur during purification.

The purified material described above was also subject to analysis forglycosylation. Purified native PAF-AH was incubated in the presence orabsence of N-Glycanase, an enzyme that removes N-linked carbohydratesfrom glycoproteins. The treated PAF-AH samples were electrophoresedthrough a 12% SDS polyacrylamide gel then visualized by Western blottingusing rabbit polyclonal antisera. Protein not treated with N-Glycanasemigrated as a diffuse band of 45-50 kDa whereas the protein treated withthe glycanase migrated as a tight band of about 44 kDa, demonstratingthat native PAF-AH is glycosylated.

N-terminal heterogeneity was also observed in purified preparations ofrecombinant PAF-AH (Ile₄₂ N-terminus). These preparations were a mixtureof polypeptides with N-termini beginning at Ala₄₇, Ile₄₂, or theartificial initiating Met₋₁ adjacent to Ile₄₂.

1. Preliminary comparison of PAF-AH fragments with PAF-AH

In view of the observed heterogeneity of recombinantly produced PAF-AH,other recombinant products were prepared and tested for homogeneityafter recombinant expression and purification. The composition of therecombinant expression products of pBAR2/PH.2 and pBAR2/PH.9 in E. colistrain MC1061 was analyzed at different time points during theproduction phase of cell fermentation. Partially purified samples of therecombinant PH.2 and PH.9 from cells collected at time points rangingbetween 5 and 22 hours after induction of protein expression wereanalyzed by matrix assisted laser desorption ionization massspectrometry (MALDI-MS).

When the PH.2 expression vector was utilized, two peaks were observed inthe spectrum of the partially purified protein at a mass value expectedfor rPAF-AH protein. Two peaks were observed at all time points, withgreater heterogeneity being observed at time points when fermentation isstressed as indicated by an accumulation of acetate and/or a depletionof oxygen in the media. The accuracy of the MALDI-MS technique in thismass range was approximately ±0.3%, about the mass of one amino acid.The higher mass peak observed was consistent with the presence of theexpected full length translation product for the PH.2 vector, minus thetranslation initiating methionine which is expected to bepost-translationally removed. The lower mass peak was approximately 1200atomic mass units less.

When the PH.9 expression vector was utilized, a single peak predominatedin the spectrum of the partially purified protein at a mass valueexpected for rPAF-AH protein. This single peak was observed at all timepoints, with no increase in heterogeneity seen at different time points.The observed mass of this protein was consistent with the presence ofthe expected full length translation product for the PH.9 vector, minusthe initiating methionine.

2. Purification of PAF-AH fragments

Recombinantly expressed rPH.2 (the expression product of DNA encodingMet₄₆ -Asn₄₄₁) and rPH.9 (the expression product of DNA encoding Met₄₆-Ile₄₂₉) preparations were purified for further comparison with purifiedrPAF-AH (expression product of DNA encoding Ile₄₂ -Asn₄₄₁). rPH.9 wasproduced by E. coli strain SB7219 and purified generally according tothe zinc chelate purification procedure described above, while rPH.2 wasproduced by E. coli strain MC1061 and purified as described below. Thetransformed cells were lysed by dilution of the cell paste with lysisbuffer (100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0). The slurrywas mixed and lysed by high pressure disruption. The lysed cells werecentrifuged and the supernatant containing rPH.2 was retained. Theclarified supernatant was diluted 5-fold in 25 mM sodium phosphatebuffer containing, 1 mM EDTA, 10 mM CHAPS, pH 7.0. The dilutedsupernatant was then applied to the Q Sepharose column. The column waswashed first with 3 column volumes of 25 mM sodium phosphate buffercontaining 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS, pH 7.0 (Wash 1), thenwashed with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA,10 mM CHAPS, pH 8.0 (Wash 2) and with 10 column volumes of 25 mM Trisbuffer containing 1 mM EDTA, 100 mM NaCl, 10 mM CHAPS, pH 8.0 (Wash 3).Elution was accomplished with 25 mM Tris buffer containing 1 mM EDTA,350 mM NaCl, 10 mM CHAPS, pH 8.0. The Q Sepharose eluate was diluted3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then applied to aBlue Sepharose column. The column was washed first with 10 columnvolumes of 25 mM Tris, 1 mM EDTA. 10 mM CHAPS, pH 8.0. The column wasthen washed with 3 column volumes of 25 mM Tris, 0.5 M NaCl, 10 mMCHAPS, pH 8.0. Elution was accomplished with 25 mM Tris, 3.0 M NaCl, 10mM CHAPs, pH 8.0. The Blue Sepharose eluate was diluted 5-fold in 10 mMsodium phosphate, 10 mM CHAPS, pH 6.2 then applied to the chromatographycolumn. The column was washed with 10 column volumes of 10 mM sodiumphosphate, 100 mM NaCl, 0.1% Pluronic F68, pH 6.2. rPH.2 was eluted with120 mM sodium phosphate, 100 mM NaCl, 0.1% Pluronic F-68, pH 7.5. Thehydroxyapatite eluate was diluted 6-fold with 10 mM sodium phosphate,0.1% Pluronic F68, pH 6.8. The diluted hydroxyapatite eluate wasadjusted to pH 6.8 using 0.5 N succinic acid and then applied to a SPSepharose column. The SP Sepharose column was washed with 10 columnvolumes 50 mM sodium phosphate, 0.1% Fluronic F68, pH 6.8 and elutedwith 50 mM sodium phosphate, 125 mM NaCl, 0.1% Pluronic F68, pH 7.5. Theeluted rPH.2 was formulated by diluting to a final concentration of 4mg/ml in 50 mM sodium phosphate, 125 mM NaCl, 0.15% Pluronic F68, pH7.5, and Tween 80 was added to a final concentration of 0.02% Tween 80.The formulated product was then filtered through a 0.2μ membrane andstored prior to use.

3. Comparison of PAF-AH fragments with PAF-AH by sequencing

The purified rPH.2 and rPH.9 preparations were compared with purifiedrPAF-AH preparations by N-terminal sequencing using an AppliedBiosystems Model 473A Protein Sequencer (Applied Biosystems, FosterCity, Calif.) and by C-terminal sequencing using a Hewlett-Packard ModelG1009A C-terminal Protein Sequencer. The rPH.2 preparation had lessN-terminal heterogeneity compared to rPAF-AH. The N-terminus analysis ofthe rPH.9 preparation was similar to that of rPH.2, but less C-terminalheterogeneity was observed for the rPH.9 preparation relative to rPH.2.

The purified rPH.2 preparation contained a major sequence with anN-terminus of Ala₄₇ (about 86-89%) and a minor sequence with anN-terminus of Ala₄₈ (about 11-14%), with the ratio of the two N-terminibeing fairly consistent under different fermentation conditions. Thepurified rPH.9 preparation also contained a major sequence with anN-terminus of Ala₄₇ (about 83-90%) and a minor sequence with anN-terminus of Ala₄₈ (about 10-17%). In contrast, attempts to produce inbacteria the polypeptide beginning at Ile₄₂ (rPAF-AH) resulted in avarying mixture of polypeptides with N-termini beginning at Ala₄₇(20-53%), Ile₄₂ (8-10%), or at the artificial initiating Met₋₁methionine (37-72%) adjacent to Ile₄₂. For rPH.2 and rPH.9, theinitiating methionine is efficiently removed by an amino-terminalpeptidase after bacterial synthesis of the polypeptide, leaving thealanine at position 47 (or the alanine at position 48) as the N-terminalresidue.

C-terminal sequencing was carried out on one lot of rPH.2, which wasobserved to have a C-terminus of HOOC-Asn-Tyr as the major sequence(about 80%), consistent with the predicted HOOC-Asn₄₄₁ -Tyr₄₄₀C-terminus of the translation product, while about 20% was HOOC-Leu.After the rPH.2 preparation had been fractionated by SDS-PAGE,additional sequencing of the primary and secondary bands yielded aC-terminal sequence of HOOC-Leu-Met from a lower secondary band (AH_(L),described below in section B.5.) consistent with a product that is 10amino acids shorter than the full length translation product, as well aslow levels of HOOC-His. Further peptide mapping has shown thatadditional C-termini are present in some lots of PH.2 protein. TheC-terminus of rPH.9 was primarily HOOC-Ile-His (about 78 to 91%,depending on the lot) by direct sequencing, consistent with thepredicted HOOC-Ile₄₂₉ -His₄₂₈ C-terminus of the translation product.There appears to be some background ("noise") in this technique, so lowlevels of other sequences could not be ruled out.

4. Comparison of PAF-AH fragments with PAF-AH by MALDI-MS

MALDI-MS was performed on purified rPH.2 and rPH.9 preparations. TherPH.2 spectrum exhibited two peaks in the spectrum at a mass valueexpected for the rPAF-AH product (see FIG. 4), similar to the patternobserved with the partially purified protein in section B.1. above. Thesecondary, lower molecular weight peak was typically present atapproximately 20% to 30% of the total. The rPH.9 spectrum showed apredominant peak at a mass consistent with that expected for the fulllength translation product for the PH.9 vector, minus the translationinitiating methionine (see FIG. 5). A small slightly lower molecularweight shoulder peak was also observed for rPH.9 that representedapproximately 5% of the total.

5. Comparison of PAF-AH fragments with PAF-AH by SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) wasperformed on purified rPAF-AH, rPH.2 and rPH.9 preparations. Acomplicated banding pattern was observed for rPH.2 around theelectrophoretic migration range expected for the rPAF-AH product, basedon protein molecular weight standards. One, or in some gels, twopredominant bands were seen, with readily observed secondary bands aboveand below the primary band. These upper secondary, middle primary andlower secondary bands, respectively, were termed AH_(U), AH_(M) andAH_(L). All of these bands reacted with an anti-rPAF-AH monoclonalantibody on Western blot and have thus been identified as rPAF-AHrelated products. The upper secondary band AH_(U) increased in intensityover time with storage of the protein and presumably represents amodified form of the rPAF-AH product. The SDS-PAGE of the rPAF-AHpreparation is similar to that of rPH.2. There are two major bands thatmigrate near the expected molecular weight for rPAF-AH, as well as aminor band above and a shadow below the major bands. In contrast, rPH.9displayed a single predominant band on SDS-PAGE with no apparentsplitting. Faint bands at a slightly lower molecular weight and at anexpected dimer position were also seen. No AH_(U) -like band wasobserved.

The composition of the purified rPH.2 and rPH.9 preparations was alsoanalyzed on 2D gels (isoelectric focusing (I) in urea followed bySDS-PAGE in the second dimension). For rPH.9, the 2D gels showed fivemain spots separated in the IEF direction. The charge heterogeneityappeared consistent between lots of rPH-9. In contrast, the 2D gelpattern of rPH.2 was more complicated as it contained approximately 15spots separated in the IEF and SDS-PAGE dimensions.

6. Comparison of activity of PAF-AH fragments with PAF-AH

Purified rPH.2 and rPH.9 have enzymatic activity indistinguishable fromthat of endogenous PAF-AH purified from serum, and rPH.2 and rPH.9 bindto lipropotein in a similar manner as purified endogenous PAF-AH.

EXAMPLE 11

A preliminary analysis of expression patterns of human plasma PAF-AHmRNA in human tissues was conducted by Northern blot hybridization.

RNA was prepared from human cerebral cortex, heart, kidney, placenta,thymus and tonsil using RNA Stat 60 (Tel-Test "B", Friendswood, Tex.).Additionally, RNA was prepared from the human hematopoieticprecursor-like cell line, THP-1 (ATCC TIB 202), which was induced todifferentiate to a macrophage-like phenotype using the phorbol esterphorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from thepremyelocytic THP-1 cell line prior to and 1 to 3 days after inductionwere electrophoresed through a 1.2% agarose formaldehyde gel andsubsequently transferred to a nitrocellulose membrane. The full lengthhuman plasma PAF-AH cDNA, sAH 406-3, was labelled by random priming andhybridized to the membrane under conditions identical to those describedin Example 3 for library screening. Initial results indicate that thePAF-AH probe hybridized to a 1.8 kb band in the thymus, tonsil, and to alesser extent, the placental RNA.

PAF is synthesized in the brain under normal physiological as well aspathophysiological conditions. Given the known pro-inflammatory andpotential neurotoxic properties of the molecule, a mechanism forlocalization of PAF synthesis or for its rapid catabolism would beexpected to be critical for the health of neural tissue. The presence ofPAF acetylhydrolase in neural tissues is consistent with it playing sucha protective role. Interestingly, both a bovine heterotrimericintracellular PAF-AH [the cloning of which is described in Hattori etal., J. Biol. Chem., 269(37): 23150-23155 (1994)] and PAF-AH of theinvention have been identified in the brain. To determine whether thetwo enzymes are expressed in similar or different compartments of thebrain, the human homologue of the bovine brain intracellular PAF-AH cDNAwas cloned, and its mRNA expression pattern in the brain was compared byNorthern blotting to the mRNA expression pattern of the PAF-AH of theinvention by essentially the same methods as described in the foregoingparagraph. The regions of the brain examined by Northern blotting werethe cerebellum, medulla, spinal cord, putamen, amygdala, caudatenucleus, thalamus, and the occipital pole, frontal lobe and temporallobe of the cerebral cortex. Message of both enzymes was detected ineach of these tissues although the heterotrimeric intracellular formappeared in greater abundance than the secreted form. Northern blotanalysis of additional tissues further revealed that the heterotrimericintracellular form is expressed in a broad variety of tissues and cells,including thymus, prostate, testis, ovary, small intestine, colon,peripheral blood leukocytes, macrophages, brain, liver, skeletal muscle,kidney, pancreas and adrenal gland. This ubiquitous expression suggeststhat the heterotrimeric intracellular PAF-AH has a general housekeepingfunction within cells.

The expression of PAF-AH RNA in monocytes isolated from human blood andduring their spontaneous differentiation into macrophages in culture wasalso examined. Little or no RNA was detected in fresh monocytes, butexpression was induced and maintained during differentiation intomacrophages. There was a concomitant accumulation of PAF-AH activity inthe culture medium of the differentiating cells. Expression of the humanplasma PAF-AH transcript was also observed in the THP-1 cell RNA at 1day but not 3 days following induction. THP-1 cells did not express mRNAfor PAF-AH in the basal state.

EXAMPLE 12

PAF-AH expression in human and mouse tissues was examined by in situhybridization.

Human tissues were obtained from National Disease Research Interchangeand the Cooperative Human Tissue Network. Normal mouse brain and spinalcord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice.Normal S/JLJ mouse embryos were harvested from eleven to eighteen daysafter fertilization.

The tissue sections were placed in Tissue Tek II cryomolds (MilesLaboratories, Inc., Naperville, Ill.) with a small amount of OCTcompound (Miles, Inc., Elkhart, Ind.). They were centered in thecryomold, the cryomold filled with OCT compound, then placed in acontainer with 2-methylbutane [C₂ H₅ CH(CH₃)₂, Aldrich Chemical Company,Inc., Milwaukee, Wis.] and the container placed in liquid nitrogen. Oncethe tissue and OCT compound in the cryomold were frozen, the blocks werestored at -80° C. until sectioning. The tissue blocks were sectioned at6 μm thickness and adhered to Vectabond (Vector Laboratories, Inc.,Burlingame, Calif.) coated slides and stored at -70° C. and placed at50° C. for approximately 5 minutes to warm them and remove condensationand were then fixed in 4% paraformaldehyde for 20 minutes at 4° C.,dehydrated (70%, 95%, 100% ethanol) for 1 minute at 4° C. in each grade,then allowed to air dry for 30 minutes at room temperature. Sectionswere denatured for 2 minutes at 70° C. in 70% formamide/2×SSC, rinsedtwice in 2×SSC, dehydrated and then air dried for 30 minutes. Thetissues were hybridized in situ with radiolabeled single-stranded mRNAgenerated from DNA derived from an internal 1 Kb HindIII fragment of thePAF-AH gene (nucleotides 308 to 1323 of SEQ ID NO: 7) by in vitro RNAtranscription incorporation ³⁵ S-UTP (Amersham) or from DNA derived fromthe heterotrimeric intracellular PAF-AH cDNA identified by Hattori etal. The probes were used at varying lengths from 250-500 bp.Hybridization was carried out overnight (12-16 hours) at 50° C.; the ³⁵S-labeled riboprobes (6×10⁵ cpm/section), tRNA (0.5 μg/section) anddiethylpyrocarbonate (depc)-treated water were added to hybridizationbuffer to bring it a final concentration of 50% formamide, 0.3M NaCl, 20mM Tris pH 7.5, 10% dextran sulfate, 1×Denhardt's solution, 100 mMdithiothretol (DTT) and 5 mM EDTA. After hybridization, sections werewashed for 1 hour at room temperature in 4×SSC/10 mM DTT, then for 40minutes at 60° C. in 50% formamide/1×SSC/10 mM DTT, 30 minutes at roomtemperature in 2×SSC, and 30 minutes at room temperature in 0.1×SSC. Thesections were dehydrated, air dried for 2 hours, coated with Kodak NTB2photographic emulsion, air dried for 2 hours, developed (after storageat 4° C. in complete darkness) and counterstained withhematoxylin/eosin.

A. Brain

Cerebellum. In both the mouse and the human brains, strong signal wasseen in the Purkinje cell layer of the cerebellum, in basket cells, andindividual neuronal cell bodies in the dentate nucleus (one of the fourdeep nuclei in the cerebellum). Message for the heterotrimericintracellular PAF-AH was also observed in these cell types.Additionally, plasma PAF-AH signal was seen on individual cells in thegranular and molecular layers of the grey matter.

Hippocampus. In the human hippocampus section, individual cellsthroughout the section, which appear to be neuronal cell bodies, showedstrong signal. These were identified as polymorphic cell bodies andgranule cells. Message for the heterotrimeric intracellular PAF-AH wasalso observed in hippocampus.

Brain stem. On both human and mouse brain stem sections, there wasstrong signal on individual cells in the grey matter.

Cortex. On human cortex sections taken from the cerebral, occipital, andtemporal cortexes, and on mouse whole brain sections, individual cellsthroughout the cortex showed strong signal. These cells were identifiedas pyramidal, stellate and polymorphic cell bodies. There does notappear to be differentiation in the expression pattern in the differentlayers of the cortex. These in situ hybridization results are differentfrom the results for cerebral cortex obtained by Northern blotting. Thedifference is likely to result from the greater sensitivity of in situhybridization compared to that of Northern blotting. As in thecerebellum and hippocampus, a similar pattern of expression of theheterotrimeric intracellular PAF-AH was observed.

Pituitary. Somewhat weak signal was seen on scattered individual cellsin the pars distalis of the human tissue section.

B. Human colon

Both normal and Crohn's disease colons displayed signal in the lymphaticaggregations present in the mucosa of the sections, with the level ofsignal being slightly higher in the section from the Crohn's diseasepatient. The Crohn's disease colon also had strong signal in the laminapropria. Similarly, a high level of signal was observed in a diseasedappendix section while the normal appendix exhibited a lower but stilldetectable signal. The sections from the ulcerative colitis patientshowed no evident signal in either the lymphatic aggregations or thelamina propria.

C. Human tonsil and thymus

Strong signal was seen on scattered groups of individual cells withinthe germinal centers of the tonsil and within the thymus.

D. Human lymph node

Strong signal was observed on the lymph node section taken from a normaldonor, while somewhat weak signal was observed in the lymph nodules ofthe section from a donor with septic shock.

E. Human small intestine

Both normal and Crohn's disease small intestine had weak signal in thePeyer's patches and lamina propria in the sections, with the signal onthe diseased tissue slightly higher.

F. Human spleen and lung

Signal was not observed on any of the spleen (normal and splenic abcesssections) or lung (normal and emphysema sections) tissues.

G. Mouse spinal cord

In both the normal and EAE stage 3 spinal cords, there was strong signalin the grey matter of the spinal cord, with the expression beingslightly higher in the EAE stage 3 spinal cord. In the EAE stage 3spinal cord, cells in the white matter and perivascular cuffs, probablyinfiltrating macrophages and/or other leukocytes, showed signal whichwas absent in the normal spinal cord.

F. Mouse embryos

In the day 11 embryo signal was apparent in the central nervous systemin the fourth ventricle, which remained constant throughout the embryotime course as it developed into the cerebellum and brain stem. As theembryos matured, signal became apparent in central nervous system in thespinal cord (day 12), primary cortex and ganglion Gasseri (day 14), andhypophysis (day 16). Signal was observed in the peripheral nervoussystem (beginning on day 14 or 15) on nerves leaving the spinal cord,and, on day 17, strong signal appeared around the whiskers of theembryo. Expression was also seen in the liver and lung at day 14, thegut (beginning on day 15), and in the posterior portion of themouth/throat (beginning on day 16). By day 18, the expression patternhad differentiated into signal in the cortex, hindbrain (cerebellum andbrain stem), nerves leaving the lumbar region of the spinal cord, theposterior portion of the mouth/throat, the liver, the kidney, andpossible weak signal in the lung and gut.

G. Summary

PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer'spatches, appendix, and colon lymphatic aggregates is consistent with theconclusions that the probable predominant in vivo source of PAF-AH isthe macrophage because these tisues all are populated with tissuemacrophages that serve as phagocytic and antigen-processing cells.

Expression of PAF-AH in inflamed tissues would be consistent with thehypothesis that a role of monocyte-derived macrophages is to resolveinflammation. PAF-AH would be expected to inactivate PAF and thepro-inflammatory phospholipids, thus down-regulating the inflammatorycascade of events initiated by these mediators.

PAF has been detected in whole brain tissue and is secreted by ratcerebellar granule cells in culture. In vitro and in vivo experimentshave demonstrated that PAF binds a specific receptor in neural tissuesand induces functional and phenotypic changes such as calciummobilization, upregulation of transcription activating genes, anddifferentiation of the neural precursor cell line, PC12. Theseobservations suggested a physiologic role for PAF in the brain, andconsistent with this, recent experiments using hippocampal tissuesection cultures and PAF analogs and antagonists have implicated PAF asan important retrograde messenger in hippocampal long term potentiation.Therefore, in addition to its pathological effect in inflammation, PAFappears to participate in routine neuronal signalling processes.Expression of the extracellular PAF-AH in the brain may serve toregulate the duration and magnitude of PAF-mediated signalling.

EXAMPLE 13

Monoclonal antibodies specific for recombinant human plasma PAF-AH weregenerated using E. coli produced PAF-AH as an immunogen.

Mouse #1342 was injected on day 0, day 19, and day 40 with recombinantPAF-AH. For the prefusion boost, the mouse was injected with theimmunogen in PBS, four days later the mouse was sacrificed and itsspleen removed sterilely and placed in 10 ml serum free RPMI 1640. Asingle-cell suspension was formed by grinding the spleen between thefrosted ends of two glass microscope slides submerged in serum free RPMI1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).The cell suspension was filtered through sterile 70-mesh Nitex cellstrainer (Becton Dickinson, Parsippany, N.J.), and washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 20 mlserum free RPMI. Thymocytes taken from 3 naive Balb/c mice were preparedin a similar manner. NS-1 myeloma cells, kept in log phase in RPMI with11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah)for three days prior to fusion, were centrifuged at 200 g for 5 minutes,and the pellet was washed twice as described in the foregoing paragraph.

One×10⁸ spleen cells were combined with 2.0×10⁷ NS-1 cells, centrifugedand the supernatant was aspirated. The cell pellet was dislodged bytapping the tube and 1 ml of 37° C. PEG 1500 (50% in 75 mM Hepes, pH8.0) (Boehringer Mannheim) was added with stirring over the course of 1minute, followed by adding 7 ml of serum free RPMI over 7 minutes. Anadditional 8 ml RPMI was added and the cells were centrifuged at 200 gfor 10 minutes. After discarding the supernatant, the pellet wasresuspended in 200 ml RPMI containing 15% FBS, 100 μM sodiumhypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/ml and platedinto 10 Corning flat bottom 96 well tissue culture plates (Corning,Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium was removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion was screened by ELISA, testing for the presence of mouse IgGbinding to recombinant PAF-AH. Immulon 4 plates (Dynatech, Cambridge,Mass.) were coated for 2 hours at 37° C. with 100 ng/well recombinantPAF-AH diluted in 25 mM TRIS, pH 7.5. The coating solution was aspiratedand 200 ul/well of blocking solution [0.5% fish skin gelatin (Sigma)diluted in CMF-PBS] was added and incubated for 30 minutes at 37° C.Plates were washed three times with PBS with 0.05% Tween 20 (PBST) and50 μl culture supernatant was added. After incubation at 37° C. for 30minutes, and washing as above, 50 μl of horseradish peroxidaseconjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove,Pa.) diluted 1:3500 in PBST was added. Plates were incubated as above,washed four times with PBST and 100 μL substrate, consisting of 1 mg/mlo-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂ O₂ in 100 mM Citrate,pH 4.5, was added. The color reaction was stopped in 5 minutes with theaddition of 50 μl of 15% H₂ SO₄. A₄₉₀ was read onn a plate reader(Dynatech).

Selected fusion wells were cloned twice by dilution into 96 well platesand visually scoring the number of colonies/well after 5 days.Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and90F2D (ATCC HB 11725).

The monoclonal antibodies produced by hybridomas were isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.). Resultsshowed that the monoclonal antibodies produced by hybridomas from fusion90 were all IgG₁.

All of the monoclonal antibodies produced by hybridomas from fusion 90functioned well in ELISA assays but were unable to bind PAF-AH onWestern blots. To generate antibodies that could recognize PAF-AH byWestern, mouse #1958 was immunized with recombinant enzyme. Hybridomaswere generated as described for fusion 90 but were screened by Westernblotting rather than ELISA to identify Western-competent clones.

For Western analyses, recombinant PAF-AH was mixed with an equal volumeof sample buffer containing 125 mM Tris, pH 6.8, 4% SDS, 100 mMdithiothreitol and 0.05% bromphenol blue and boiled for five minutesprior to loading onto a 12% SDS polyacrylamide gel (Novex). Followingelectrophoresis at 40 mAmps, proteins were electrotransferred onto apolyvinylidene fluoride membrane (Pierce) for 1 hour at 125 V in 192 mMglycine, 25 mM Tris base, 20% methanol, and 0.01% SDS. The membrane wasincubated in 20 mM Tris, 100 mM NaCl (TBS) containing 5% bovine serumalbumin (BSA, Sigma) overnight at 4° C. The blot was incubated 1 hour atroom temperature with rabbit polyclonal antisera diluted 1/8000 in TBScontaining 5% BSA, and then washed with TBS and incubated with alkalinephosphatase-conjugated goat anti-mouse IgG in TBS containing 5% BSA for1 hour at room temperature. The blot was again washed with TBS thenincubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03%nitroblue tetrazolium in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mMMgCl₂. The reaction was stopped with repeated water rinses.

Selected fusion wells, the supernatants of which were positive inWestern analyses, were processed as described above. Hybridoma 143Areacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).

Polyclonal antisera specific for human plasma PAF-AH was raised inrabbits by three monthly immunizations with 100 μg of purifiedrecombinant enzyme in Fruend's adjuvant.

EXAMPLE 14

Experimental studies were performed to evaluate the in vivo therapeuticeffects of recombinant PAF-AH of the invention on acute inflammationusing a rat foot edema model [Henriques et al., Br. J. Pharmacol., 106:579-582 (1992)]. The results of these studies demonstrated that rPAF-AHblocks PAF-induced edema. Parallel studies were done to compare theeffectiveness of PAF-AH with two commercially available PAF antagonists.

A. Preparation of PAF-AH

E. coli transformed with the PAF-AH expression vector puc trp AH werelysed in a microfluidizer, solids were centrifuged out and the cellsupernatants were loaded onto a S-Sepharose column (Pharmacia). Thecolumn was washed extensively with buffer consisting of 50 mM NaCl, 10mM CHAPS, 25 mM MES and 1 mM EDTA, pH 5.5. PAF-AH was eluted byincreasing the NaCl concentration of the buffer to 1M. Affinitychromatography using a Blue Sepharose column (Pharmacia) was then usedas an additional purification step. Prior to loading the PAF-AHpreparation on the Blue Sepharose column, the sample was diluted 1:2 toreduce the NaCl concentration to 0.5M and the pH was adjusted to 7.5.After washing the Blue Sepharose column extensively with bufferconsisting of 0.5M NaCl, 25 mM tris, 10 mM CHAPS and 1 mM EDTA, pH 7.5the PAF-AH was eluted by increasing the NaCl concentration to 3.0M.

Purity of PAF-AH isolated in this manner was generally 95% as assessedby SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additionalquality controls done on each PAF-AH preparation included determiningendotoxin levels and hemolysis activity on freshly obtained raterythrocytes. A buffer containing 25 mM Tris, 10 mM CHAPS, 0.5M NaCl, pH7.5 functioned as storage media of the enzyme as well as carrier foradministration. Dosages used in experiments were based on enzymeactivity assays conducted immediately prior to experiments.

B. Induction of Edema

Six to eight-week-old female Long Evans rats (Charles River, Wilmington,Mass.), weighing 180-200 grams, were used for all experiments. Prior toexperimental manipulations, animals were anesthetized with a mixture ofthe anesthetics Ketaset (Fort Dodge Laboratories, Fort Dodge, Iowa),Rompun (Miles, Shawnee Mission, Kans.), and Ace Promazine (Aveco, FortDodge, Iowa) administered subcutaneously at approximately 2.5 mgKetaset, 1.6 mg Rompun, 0.2 mg Ace Promazine per animal per dose. Edemawas induced in the foot by administration of either PAF or zymosan asfollows. PAF (Sigma #P-1402) was freshly prepared for each experimentfrom a 19.1 mM stock solution stored in chloroform/methanol (9:1) at-20° C. Required volumes were dried down under N₂, diluted 1:1000 in abuffer containing 150 mM NaCl, 10 mM Tris pH 7.5, and 0.25% BSA, andsonicated for five minutes. Animals received 50 μl PAF (final dose of0.96 nmoles) subcutaneously between the hind foot pads, and edema wasassessed after 1 hour and again after 2 hours in some experiments.Zymosan A (Sigma #A-8800) was freshly prepared for each experiment as asuspension of 10 mg/ml in PBS. Animals received 50 μl of zymosan (finaldose of 500 μg) subcutaneously between the hind foot pads and edema wasassessed after 2 hours.

Edema was quantitated by measuring the foot volume immediately prior toadministration of PAF or zymosan and at indicated time pointpost-challenge with PAF or zymosan. Edema is expressed as the increasein foot volume in milliliters. Volume displacement measurements weremade on anesthetized animals using a plethysmometer (UGO Basile, model#7150) which measures the displaced water volume of the immersed foot.In order to insure that foot immersion was comparable from one timepoint to the next, the hind feet were marked in indelible ink where thehairline meets the heel. Repeated measurements of the same foot usingthis technique indicate the precision to be within 5%.

C. PAF-AH Administration Routes and Dosages

PAF-AH was injected locally between the foot pads, or systematically byIV injection in the tail vein. For local administration rats received100 μl PAF-AH (4000-6000 U/ml) delivered subcutaneously between theright hind foot pads. Left feet served as controls by administration of100 μl carrier (buffered salt solution). For systemic administration ofPAF-AH, rats received the indicated units of PAF-AH in 300 μl of carrieradministered IV in the tail vein. Controls received the appropriatevolume of carrier IV in the tail vein.

D. Local Administration of PAF-AH

Rats (N=4) were injected with 100 μl of PAF-AH (4000-6000 U/ml)subcutaneously between the right foot pads. Left feet were injected with100 μl carrier (buffered salt solution). Four other rats were injectedonly with carrier. All rats were immediately challenged with PAF viasubcutaneous foot injection and foot volumes assessed 1 hourpost-challenge. FIG. 6, wherein edema is expressed as average increasein foot volume (ml)±SEM for each treatment group, illustrates thatPAF-induced foot edema is blocked by local administration of PAF-AH. Thegroup which received local PAF-AH treatment prior to PAF challengeshowed reduced inflammation compared to the control injected group. Anincrease in foot volume of 0.08 ml±0.08 (SEM) was seen in the PAF-AHgroup as compared to 0.63±0.14 (SEM) for the carrier treated controls.The increase in foot volume was a direct result of PAF injection asanimals injected in the foot only with carrier did not exhibit anincrease in foot volume.

E. Intravenous Administration of PAF-AH

Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in300 μl carrier) or carrier alone, 15 minutes prior to PAF challenge.Edema was assessed 1 and 2 hours after PAF challenge. FIG. 7, whereinedema is expressed as average increase in volume (ml)±SEM for eachtreatment group, illustrates that IV administration of PAF-AH blockedPAF induced foot edema at one and two hours post challenge. The groupwhich received 2000 U of PAF-AH given by the IV route showed a reductionin inflammation over the two hour time course. Mean volume increase forthe PAF-AH treated group at two hours was 0.10 ml±0.08 (SEM), versus0.56 ml±0.11 for carrier treated controls.

F. Comparison of PAF-AH Protection in Edema Induced by PAF or Zymosan

Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in300 μl carrier) or carrier alone. Fifteen minutes after pretreatment,groups received either PAF or zymosan A, and foot volume was assessedafter 1 and 2 hours, respectively. As shown in FIG. 8, wherein edema isexpressed as average increase in volume (ml)±SEM for each treatmentgroup, systemic administration of PAF-AH (2000 U) was effective inreducing PAF-induced foot edema, but failed to block zymosan inducededema. A mean increase in volume of 0.08±0.02 was seen in the PAF-AHtreated group versus 0.49±0.03 for the control group.

G. Effective Dose Titration of PAF-AH Protection

In two separate experiments, groups of rats (N=3 to 4 per group) werepretreated IV with either serial dilutions of PAF-AH or carrier controlin a 300 μl volume, 15 minutes prior to PAF challenge. Both feet werechallenged with PAF (as described above) and edema was assessed after 1hour. FIG. 9 wherein edema is expressed as average increase in volume(ml)±SEM for each treatment group, illustrates the increase inprotection from PAF-induced edema in rats injected with increasingdosages of PAF-AH. In the experiments, the ID₅₀ of PAF-AH given by theIV route was found to be between 40 and 80 U per rat.

H. In Vivo Efficacy of PAF-AH as a Function of Time After Administration

In two separate experiments, two groups of rats (N=3 to 4 per group)were pretreated IV with either PAF-AH (2000 U in 300 μl carrier) orcarrier alone. After administration, groups received PAF at time pointsranging from 15 minutes to 47 hours post PAF-AH administration. Edemawas then assessed 1 hour after PAF challenge. As shown in FIG. 10,wherein edema is expressed as average increase in volume (ml)±SEM foreach treatment group, administration of 2000 U of PAF-AH protects ratsfrom PAF induced edema for at least 24 hours.

I. Pharmacokinetics of PAF-AH

Four rats received 2000 U of PAF-AH by IV injection in a 300 μl volume.Plasma was collected at various time points and stored at 4° C. andplasma concentrations of PAF-AH were determined by ELISA using a doublemAb capture assay. In brief, monoclonal antibody 90G11D (Example 13) wasdiluted in 50 mM carbonate buffer pH 9.6 at 100 ng/ml and immobilized onImmulon 4 ELISA plates overnight at 4° C. After extensive washing withPBS containing 0.05% Tween 20, the plates were blocked for 1 hour atroom temperature with 0.5% fish skin gelatin (Sigma) diluted in PBS.Serum samples diluted in PBS with 15 mM CHAPS were added in duplicate tothe washed ELISA plate and incubated for 1 hour at room temperature.After washing, a biotin conjugate of monoclonal antibody 90F2D (Example13) was added to the wells at a concentration of 5 μg/ml diluted in PBSand incubated for 1 hour at room temperature. After washing, 50 μl of a1:1000 dilution of ExtraAvidin (Sigma) was added to the wells andincubated for 1 hour at room temperature. After washing, wells weredeveloped using OPD as a substrate and quantitated. Enzyme activity wasthen calculated from a standard curve. FIG. 11, wherein data pointsrepresent means±SEM, shows that at one hour plasma enzyme levelsapproached the predicted concentration based on a 5-6 ml plasma volumefor 180-200 gram rats, mean=374 U/ml±58.2. Beyond one hour plasma levelssteadily declined, reaching a mean plasma concentration of 19.3 U/ml±3.4at 24 hours, which is still considerably higher than endogenous ratPAF-AH levels which have been found to be approximately 4 U/ml byenzymatic assays.

J. Effectiveness of PAF-AH Versus PAF Antagonists

Groups of rats (N=4 per group) were pretreated with one of threepotential antiinflammatories: the PAF antagonist CV3988 (Biomol #L-103)administered IP (2 mg in 200 μl EtOH), the PAF antagonist Alprazolam(Sigma #A-8800) administered IP (2 mg in 200 μl EtOH), or PAF-AH (2000U) administered IV. Control rats were injected IV with a 300 μl volumeof carrier. The PAF antagonists were administered IP because they aresolubilized in ethanol. Rats injected with either CV3988 or Alprazolamwere challenged with PAF 30 minutes after administration of the PAFantagonist to allow the PAF antagonist to enter circulation, whilePAF-AH and carrier-treated rats were challenged 15 minutes after enzymeadministration. Rats injected with PAF-AH exhibited a reduction inPAF-induced edema beyond that afforded by the established PAFantagonists CV3988 and Alprazolam. See FIG. 12 wherein edema isexpressed as average increase in volume (ml)±SEM for each treatmentgroup.

In summary, rPAF-AH is effective in blocking edema mediated by PAF invivo. Administration of PAF-AH products can be either local or systemicby IV injection. In dosing studies, IV injections in the range of160-2000 U/rat were found to dramatically reduce PAF mediatedinflammation, while the ID₅₀ dosage appears to be in the range of 40-80U/rat. Calculations based on the plasma volume for 180-200 gram ratspredicts that a plasma concentration in the range of 25-40 U/ml shouldblock PAF-elicited edema. These predictions are supported by preliminarypharmacokinetic studies. A dosage of 2000 U of PAF-AH was found to beeffective in blocking PAF mediated edema for at least 24 hours. At 24hours following administration of PAF-AH plasma concentrations of theenzyme were found to be approximately 25 U/ml. PAF-AH was found to blockPAF-induced edema more effectively than the two known PAF antagoniststested.

Collectively, these results demonstrate that PAF-AH effectively blocksPAF induced inflammation and may be of therapeutic value in diseaseswhere PAF is the primary mediator.

EXAMPLE 15

Recombinant PAF-AH of the invention was tested in a second in vivomodel, PAF-induced pleurisy. PAF has previously been shown to inducevascular leakage when introduced into the pleural space [Henriques etal., supra]. Female rats (Charles River, 180-200 g) were injected in thetail vein with 200 μl of 1% Evans blue dye in 0.9% with 300 μlrecombinant PAF-AH (1500 μmol/ml/hour, prepared as described in Example14) or with an equivalent volume of control buffer. Fifteen minuteslater the rats received an 100 μl injection of PAF (2.0 nmol) into thepleural space. One hour following PAF challenge, rats were sacrificedand the pleural fluid was collected by rinsing the cavity with 3 mlheparinized phosphate buffered saline. The degree of vascular leak wasdetermined by the quantity of Evans blue dye in the pleural space whichwas quantitated by absorbance at 620 nm. Rats pretreated with PAF-AHwere found to have much less vascular leakage than control animals(representing more than an 80% reduction in inflammation).

The foregoing results support the treatment of subjects suffering frompleurisy with recombinant PAF-AH enzyme of the invention.

EXAMPLE 16

Recombinant PAF-AH enzyme of the invention was also tested for efficacyin a model of antigen-induced eosinophil recruitment. The accumulationof eosinophils in the airway is a characteristic feature of late phaseimmune responses which occur in asthma, rhinitis and eczema. BALB/c mice(Charles River) were sensitized by two intraperitoneal injectionsconsisting of 1 μg of ovalbumin (OVA) in 4 mg of aluminum hydroxide(Imject alum, Pierce Laboratories, Rockford, Ill.) given at a 2 weekinterval. Fourteen days following the second immunization, thesensitized mice were challenged with either aerosolized OVA or saline asa control.

Prior to challenge mice were randomly placed into four groups, with fourmice/group. Mice in groups 1 and 3 were pretreated with 140 μl ofcontrol buffer consisting of 25 mM tris, 0.5M NaCl, 1 mM EDTA and 0.1%Tween 80 given by intravenous injection. Mice in groups 2 and 4 werepretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in140 μl of PAF-AH buffer). Thirty minutes following administration ofPAF-AH or buffer, mice in groups 1 and 2 were exposed to aerosolized PBSas described below, while mice in groups 3 and 4 were exposed toaerosolized OVA. Twenty-four hours later mice were treated a second timewith either 140 μl of buffer (groups 1 and 3) or 750 units of PAF-AH in140 μl of buffer (groups 2 and 4) given by intravenous injection.

Eosinophil infiltration of the trachea was induced in the sensitizedmice by exposing the animals to aerosolized OVA. Sensitized mice wereplaced in 50 ml conical centrifuge tubes (Corning) and forced to breathaerosolized OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes usinga nebulizer (Model 646, DeVilbiss Corp., Somerset, Pa.). Control micewere treated in a similar manner with the exception that 0.9% saline wasused in the nebulizer. Forty-eight hours following the exposure toaerosolized OVA or saline, mice were sacrificed and the tracheas wereexcised. Tracheas from each group were inbeded in OCT and stored at -70°until sections were cut.

To evaluate eosinophil infiltration of the trachea, tissue sections fromthe four groups of mice were stained with either Luna solution andhematoxylin-eosin solution or with peroxidase. Twelve 6 μm thicksections were cut from each group of mice and numbered accordingly. Oddnumbered sections were stained with Luna stain as follows. Sections werefixed in formal-alcohol for 5 minutes at room temperature, rinsed acrossthree changes of tap water for 2 minutes at room temperature then rinsedin two changed of dH₂ O for 1 minute at room temperature. Tissuesections were stained with Luna stain 5 minutes at room temperature(Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of1% Biebrich Scarlet). Stained slides were dipped in 1% acid alcohol sixtimes, rinsed in tap water for 1 minute at room temperature, dipped in0.5% lithium carbonate solution five times and rinsed in running tapwater for 2 minutes at room temperature. Slides were dehydrated across70%-95%-100% ethanol 1 minute each, at room temperature, then cleared intwo changes of xylene for 1 minute at room temperature and mounted inCytoseal 60.

For the peroxidase stain, even numbered sections were fixed in 4° C.acetone for 10 minutes and allowed to air dry. Two hundred μl of DABsolution was added to each section and allowed to sit 5 minutes at roomtemperature. Slides were rinsed in tap water for 5 minutes at roomtemperature and 2 drops of 1% osmic acid was applied to each section for3-5 seconds. Slides were rinsed in tap water for 5 minutes at roomtemperature and counterstained with Mayers hematoxylin at 25° C. at roomtemperature. Slides were then rinsed in running tap water for 5 minutesand dehydrated across 70%-95%-100% ethanol 1 minute each at roomtemperature. Slides were cleared through two changes of xylene for 1minute each at room temperature and mounted in Cytoseal 60.

The number of eosinophils in the submucosal tissue of the trachea wasevaluated. Trachea from mice from groups 1 and 2 were found to have veryfew eosinophils scattered throughout the submucosal tissue. As expectedtracheas from mice in group 3, which were pretreated with buffer andexposed to nebulized OVA, were found to have large numbers ofeosinophils throughout the submucosal tissue. In contrast, the tracheasfrom mice in group 4, which were pretreated with PAF-AH and exposed tonebulized OVA were found to have very few eosinophils in the submucosaltissue comparable to what was seen in the two control groups, groups 1and 2.

Thus, therapeutic treatment with PAF-AH of subjects exhibiting a latephase immune response involving the accumulation of eosinophils in theairway, such as that which occurs in asthma and rhinitis is indicated.

EXAMPLE 17

A PAF-AH product of the invention was also tested in two different ratmodels for treatment of necrotizing enterocolitis (NEC), an acutehemorrhagic necrosis of the bowel which occurs in low birth weightinfants and causes a significant morbidity and mortality. Previousexperiments have demonstrated that treatment with glucocorticoidsdecreases the incidence of NEC in animals and in premature infants, andthe activity of glucocorticoids has been suggested to occur via anincrease in the activity of plasma PAF-AH.

A. Activity in Rats With NEC Induced by PAF Challenge

1. Prevention of NEC

A recombinant PAF-AH product, rPH.2 (25,500 units in 0.3 ml, groups 2and 4), or vehicle/buffer alone (25 mM tris, 0.5M NaCl, 1 mM EDTA and0.1% Tween 80) (groups 1 and 3) was administered into the tail veins offemale Wistar rats (n=3) weighing 180-220 grams. Either BSA(0.25%)-saline (groups 1 and 2) or PAF (0.2 μg/100 gm) suspended in BSAsaline (groups 3 and 4) was injected into the abdominal aorta at thelevel of the superior mesenteric artery 15 minutes after rPH.2 orvehicle injection as previously described by Furukawa, et al. [J.Pediatr.Res. 34:237-241 (1993)]. The small intestines were removed after2 hours from the ligament of Trietz to the cecum, thoroughly washed withcold saline and examined grossly. Samples were obtained from microscopicexamination from the upper, middle and lower portions of the smallintestine. The tissues were fixed in buffered formalin and the sampleprocessed for microscopic examination by staining with hematoxylin andeosin. The experiment was repeated three times.

Gross findings indicated a normal appearing bowel in groups treated withthe vehicle of BSA saline. Similarly, rPH.2 injected in the absence ofPAF had no effect on the gross findings. In contrast, the injection ofPAF into the descending aorta resulted in rapid, severe discolorationand hemorrhage of the serosal surface of the bowel. A similar hemorrhagewas noted when a section of the small bowel was examined on the mucosalside and the intestine appeared to be quite necrotic. When rPH.2 wasinjected via the tail vein 15 minutes prior to the administration of PAFinto the aorta, the bowel appeared to be normal.

Upon microscopic examination, the intestine obtained from groups 1, 2and 4 demonstrated a normal villous architecture and a normal populationof cells within the lamina propria. In contrast, the group treated withPAF alone showed a full thickness necrosis and hemorrhage throughout theentire mucosa.

The plasma PAF-AH activities were also determined in the rats utilizedin the experiment described above. PAF-AH activity was determined asfollows. Prior to the tail vein injection, blood samples were obtained.Subsequently blood samples were obtained from the vena cava just priorto the injection of PAF and at the time of sacrifice. Approximately 50μl of blood was collected in heparinized capillary tubes. The plasma wasobtained following centrifugation (980×g for 5 minutes). The enzyme wasassayed as previously described by Yasuda and Johnston, Endocrinology,130:708-716 (1992).

The mean plasma PAF-AH activity of all rats prior to injection was foundto be 75.5±2.5 units (1 unit equals 1 nmoles×min⁻¹ ×ml⁻¹ plasma). Themean plasma PAF-AH activities 15 minutes following the injection of thevehicle were 75.2±2.6 units for group 1 and 76.7±3.5 units for group 3.After 15 minutes, the plasma PAF-AH activity of the animals injectedwith 25,500 units rPH.2 was 2249±341 units for group 2 and 2494±623units for group 4. The activity of groups 2 and 4 remained elevated(1855±257 units) until the time of sacrifice (21/4 hours after rPH.2injection) (Group 2=1771±308; Group 4=1939±478). These results indicatethat plasma PAF-AH activity of the rats which were injected with thevehicle alone (groups 1 and 3) did not change during the course of theexperiment. All the animals receiving the PAF injection alone developedNEC while all rats that were injected with rPH.2 followed by PAFinjection were completely protected.

2. Dose-Dependency of Prevention of NEC

In order to determine if the protection against NEC in rats was dosedependent, animals were treated with increasing doses of rPH.2 15minutes prior to PAF administration. Initially, rPH.2, ranging from 25.5to 25,500 units were administered into the tail vein of rats. PAF (0.4μg in 0.2 ml of BSA-saline) was subsequently injected into the abdominalaorta 15 minutes after the administration of rPH.2. The small intestinewas removed and examined for NEC development 2 hours after PAFadministration. Plasma PAF-AH activity was determined prior to theexogenous administration of the enzyme, and 15 minutes and 21/4 hoursafter rPH.2 administration. The results are the mean of 2-5 animals ineach group.

Gross findings indicated that all rats receiving less than 2,000 unitsof the enzyme developed NEC. Plasma PAF-AH activity in animals receivingthe lowest protective amount of enzyme (2040 units) was 363 units per mlof plasma after 15 minutes, representing a five-fold increase over basallevels. When rPH.2 was administered at less than 1,020 total units,resultant plasma enzyme activity averaged approximately 160 or less, andall animals developed NEC.

3. Duration of Protection Against NEC

In order to determine the length of time exogenous PAF-AH productafforded protection against development of NEC, rats were injected oncewith a fixed amount of the enzyme via the tail vein and subsequentlychallenged with PAF at various time points. rPH.2 (8,500 units in 0.3ml) or vehicle alone was administered into the tail vein of rats, andPAF (0.36 μg in 0.2 ml of BSA-saline) was injected into the abdominalaorta at various times after the enzyme administration. The smallintestines were removed 2 hours after the PAF injection for gross andhistological examinations in order to evaluate for NEC development.Plasma PAF-AH activities were determined at various times after enzymeadministration and two hours after PAF administration. The meanvalue±standard error for enzyme activity was determined for each group.

Results indicated that none of the rats developed NEC within the firsteight hours after injection of rPH.2, however 100% of the animalschallenged with PAF at 24 and 48 hours following injection of the enzymedeveloped NEC.

4. Reversal of NEC

In order to determine if administration of PAF-AH product was capable ofreversing development of NEC induced by PAF injection, 25,500 units ofenzyme was administered via injection into the vena cava two minutesfollowing PAF administration (0.4 μg). None of the animals developedNEC. However, when rPH.2 was administered via this route 15 minutesafter the PAF injection, all animals developed NEC, consistent with therapid time course of NEC development as induced by the administration ofPAF previously reported Furukawa et al. [supra].

The sum of these observations indicate that a relatively small(five-fold) increase in the plasma PAF-AH activity is capable ofpreventing NEC. These observations combined with previous reports thatplasma PAF-AH activity in fetal rabbits [Maki, et al.,Proc.Natl.Acad.Sci. (USA) 85:728-732 (1988)] and premature infants[Caplan, et al., J.Pediatr. 116:908-964 (1990)] has been demonstrated tobe relatively low suggests that prophylactic administration of humanrecombinant PAF-AH products to low birth weight infants may be useful intreatment of NEC.

B. Activity in a Neonatal Model of NEC

The efficacy of a PAF-AH product, rPH.2, was evaluated as follows in anNEC model in which newborn rats are stressed by formula feeding andasphyxia, two common risk factors for the disease in humans. In thismodel, approximately 70-80% of the animals develop gross and microscopicintestinal injury similar to neonatal NEC by the third day of life.Newborn rats were obtained from pregnant Sprague-Dawley rats (HarlanSprague-Dawley, Indianapolis, Ind.) that were anesthetized with CO₂ anddelivered via abdominal incision. Newborn animals were collected, dried,and maintained in a neonatal incubator during the entire experiment.

First, separate groups of animals were used to assess the dosing andabsorption characteristics of rPH.2. Normal newborn rat pups were givenone of three different enteral or intraperitoneal doses of rPH.2 (3λ,15λ, or 75λ) at time 0, and blood was collected at 1 hour, 6 hours, or24 hours later for assessment of plasma PAF-AH activity. PAF-AH activitywas measured using a substrate incubation assay [Gray et al., Nature,374:549 (1995)] and an ELISA utilizing an anti-human rPAF-AH monoclonalantibody for each sample (90F2D and 90G11D, described in Example 13).For selected samples, immunohistochemical analysis was performed usingtwo different monoclonal antibodies developed against human rPAF-AH(90F2D and 90G11D, described in Example 13). Immunohistochemistry wasdone with standard techniques using a 1:100 dilution of the antibody andovernight incubations.

Following enteral dosing of rPH.2 in normal newborn rats, there was nomeasurable plasma PAF-AH activity at any time point using either thesubstrate incubation assay or the ELISA technique. With intraperitonealadministration of rPH.2, significant circulating PAF-AH activity wasmeasurable using both methods by 1 hour after dosing, and this activitypeaked at 6 hours. Higher doses of rPH.2 (from 3 to 75λ, 10 to 250 U)resulted in higher plasma PAF-AH activity. Immunohistochemical analysisrevealed the presence of rPAF-AH product in the epithelial cells of theintestinal mucosa following enteral administration. The reactivityclustered mostly in the intestinal villi with minimal staining presentin the crypt cells. There was more staining in the ileum than jejunum,and some rPAF-AH product was immunochemically identified in portions ofcolon. There was no demonstrable staining in any control samples or inspecimens recovered from animals dosed via the intraperitoneal route.Thus, enteral administration of rPAF-AH product resulted in localmucosal epithelial accumulation of the enzyme without any measurablesystemic absorption, while, in contrast, intraperitoneal administrationof rPAF-AH product resulted in high circulating enzyme levels but nolocal mucosal accumulation.

In the NEC model, NEC was induced in newborn rats according to Caplan etal., Pediatr. Pathol., 14:1017-1028 (1994). Briefly, animals were fedwith newborn puppy formula reconstituted from powder (Esbiliac, BordenInc) every three hours via a feeding tube. The feeding volume began at0.1 ml/feed initially and advanced as tolerated to 0.4 ml/feed by the4th day of the protocol. All animals were challenged with asphyxialinsults twice daily by breathing 100% nitrogen for 50 seconds in aclosed plastic chamber followed by exposure to cold (4° C.) for 10minutes. Bowel and bladder function was stimulated with gentlemanipulation after every feeding. Animals were maintained for 96 hoursor until they showed signs of distress. Morbid animals had abdominaldistention, bloody stools, respiratory distress, cyanosis, and lethargy,and were euthanized via decapitation. After sacrifice, the intestine ofeach rat was examined grossly for signs of necrosis and thenformalin-fixed for later histological analysis. Specimens wereparaffin-embedded, sectioned with a microtome, stained with hematoxylinand eosin, and examined in a blinded fashion by two observers.Intestinal injury was scored as 1+ for epithelial cell lifting orseparation, 2+ for sloughing of epithelial cells to mid villous level,3+ for necrosis of entire villi, and 4+ for transmural necrosis.

To assess the efficacy of rPH.2, three different groups of rats weretreated with the compound via enteral delivery, intraperitoneal deliveryor both. The rPH.2 preparation had 0.8 mg/ml protein and approximately4000 Units/mg PAF-AH activity, with a <0.5 EU/mg endotoxin/proteinratio. Enterally dosed animals were given 25λ (80 U) of rPH.2 via theorogastric tube diluted into each feeding (every three hours).Intraperitoneally dosed animals were given 75λ by intraperitonealinjection twice daily. Control animals received appropriate volumes ofbuffer (20 mM NaPO₄, pH 7.4) without the rPH.2 and were studiedsimultaneously with each experimental group. Mortality and signs of NECwere evaluated for each treatment group, and differences were analyzedstatistically using Fischer's Exact test. A p-value of <0.05 wasconsidered significant. Results are shown in Table 9 below.

                  TABLE 9                                                         ______________________________________                                                            NEC   Death                                               ______________________________________                                        Control (i. p. admin.)                                                                              7/10    8/10                                            rPH.2 (240 U i. p. twice daily)                                                                     6/11    8/11                                            Control (enteral admin.)                                                                            19/26   21/26                                           rPH.2 (80 U enterally every 3 hours)                                                                6/26    7/26                                            Control (i. p. + enteral admin.)                                                                    10/17   12/17                                           rPH.2 (240 U i. p. twice daily and                                                                  3/14    7/14                                            80 U enterally every 3 hours)                                                 ______________________________________                                         Data represent cumulative results from four different experiments for i.      p. dosing, four experiments for enteral dosing, and three experiments for     i. p. + enteral dosing.                                                  

Enteral rPH.2 administration significantly reduced the incidence of bothNEC and death compared to control animals. Results from four differententerally-dosed experiments showed that pretreatment with rPH.2decreased NEC from 19/26 (control) to 6/26 (p<0.001). Intestinal injurywas variable among treated and control animals, but in most cases wascharacterized by midvillous necrosis in some segments, total villousnecrosis in other areas, occasional areas of transmural necrosis, andremaining portions of normal intestinal histology. The worst degree ofNEC in treated animals and control animals with intestinal injury wassimilar (median score 2.8 in controls vs. 2.4 in rPH.2-treated rats,p>0.05).

Intraperitoneal dosing with rPH.2 had no significant impact on NEC ordeath in this model. The onset of symptoms was similar between thisgroup and controls (40±5 hours in controls vs 36±7 hours inrPH.2-treated rats) and the degree of NEC in both groups was similar(median score 2.6 in controls vs. 2.5 in rPH.2-treated rats).

Additional experiments were done in which rats were dosed both enterallyand intraperitoneally with rPH.2 at the same doses as the singletreatment groups (25λ of rPH.2 in each feeding every three hours, plus75λ by intraperitoneal injection twice daily). Results are shown abovein Table 9. Although there were no significant differences betweentreated and control groups in the incidence of death, the rPH.2treatment significantly reduced the incidence of NEC (10/17 in controlsvs. 3/14 in rPH.2-treated rats, p=0.04). Of note, 6 out of the 7 animalswho died in the rPH.2-treated group had positive blood cultures for E.coli obtained just prior to death.

These results further support the protective role of PAF-AH products ina neonatal model of non-PAF-induced NEC. Enteral treatment with rPAF-AHproduct prevented NEC while intraperitoneal treatment at these doses hadno demonstrable effect. These findings suggest that PAF-AH productsupplementation for formula-fed premature newborns at risk for NEC mayreduce the incidence of this disease.

EXAMPLE 18

The efficacy of PAF-AH product in a guinea pig model of acuterespiratory distress syndrome (ARDS) was examined.

Platelet-activating factor (PAF) injected intravenously into guinea pigsproduces a profound lung inflammation reminiscent of early ARDS inhumans. Within minutes after intravenous administration of PAF, the lungparenchyma becomes congested with constricted bronchi and bronchioles[Lellouch-Tubiana et al., supra. Platelets and polymorphonuclearneutrophils begin to marginate and cellular aggregates are easilyidentified along arterioles of the lung [Lellouch-Tubiana, Br. J. ExpPath., 66:345-355 (1985)]. PAF infusion also damages bronchialepithelial cells which dissociate from the airway walls and accumulatein the airway lumens. This damage to airway epithelial cells isconsistent with hyaline membrane formation that occurs in humans duringthe development of ARDS. Margination of the neutrophils and platelets isquickly followed by diapedesis of these cells into the alveolar septaand alveolar spaces of the lung. Cellular infiltrates elicited by PAFare accompanied by significant vascular leakage resulting in airwayedema [Kirsch, Exp. Lung Res., 18:447-459 (1992)]. Evidence of edema isfurther supported by in vitro studies where PAF induces a dose-dependent(10-1000 ng/ml) extravasation of ¹²⁵ I labeled fibrinogen in perfusedguinea pig lungs [Basran, Br. J. Pharmacol., 77:437 (1982)].

Based on the above observations, an ARDS model in guinea pigs wasdeveloped. A cannula is placed into the jugular vein of anaesthetizedmale Hartly guinea pigs (approximately 350-400 grams) and PAF diluted ina 500 μl volume of phosphate buffered saline with 0.25% bovine serumalbumin as a carrier (PBS-BSA) is infused over a 15 minute period oftime at a total dosage ranging from 100-400 ng/kg. At various intervalsfollowing PAF infusion, animals are sacrificed and lung tissue iscollected. In guinea pigs infused with PAF, dose dependent lung damageand inflammation is clearly evident by 15 minutes and continues to bepresent at 60 minutes. Neutrophils and red blood cells are present inthe alveolar spaces of PAF treated guinea pigs but absent in control orsham infused animals. Evidence of epithelial cell damage is also evidentand reminiscent of hyaline membrane formation in human ARDS patients.Protein determinations done on bronchoalveolar lavage (BAL) samplestaken from guinea pigs infused with PAF shows a dramatic accumulation ofprotein in the inflamed lung, clear evidence of vascular leakage.

rPH.2 was found to completely protect against PAF mediated lung injuryin the guinea pig model of ARDS. Groups of guinea pigs were pretreatedwith either rPH.2 (2000 units in 500 μl) or 500 μl of the PAF-AH bufferonly. Fifteen minutes later these guinea pigs were infused with 400ng/kg PAF in a 500 μl volume, infused over a 15 minute period. Inaddition, a sham group of guinea pigs was infused with 500 μl ofPBS-BSA. At the completion of the PAF infusion the animals weresacrificed and BAL fluid was collected by lavaging the lungs 2λ with 10ml of saline containing 2 μ/ml heparin to prevent clotting. To determineprotein concentration in the BAL, samples were diluted 1:10 in salineand the OD 280 was determined. BAL fluid from sham guinea pigs was foundto have a protein concentration of 2.10±1.3 mg/ml. In sharp contrast,BAL fluid from animals infused with PAF was found to have a proteinconcentration of 12.55±1.65 mg/ml. In guinea pigs pretreated with rPH.2,BAL fluid was found to have a protein concentration of 1.13±0.25 mg/mlwhich is comparable to the sham controls and demonstrates that PAF-AHproduct completely blocks lung edema in response to PAF.

EXAMPLE 19

The efficacy of a PAF-AH product, rPH.2, was evaluated in two differentmodels of acute pancreatitis.

A. Activity in a Rat Pancreatitis Model

Male Wistar rats (200-250 g) were purchased from Charles RiverLaboratories (Wilmington, Mass.). They were housed in a climatecontrolled room at 23±2° C. with a 12 hour light/dark cycle and fedstandard laboratory chow with water ad libitum. Animals were randomlyassigned to either control or experimental groups. Rats wereanesthetized with 50 mg/kg pentobarbital sodium intraperitoneally, and apolyvinyl catheter (size V3, Biolab products, Lake Havasu, Ariz.) wasplaced by cutdown into the jugular vein. The catheter was tunneledsubcutaneously to exit in the dorsal cervical area, and the animals wereallowed to recover from anesthesia. The rats were given free access towater but were fasted overnight. Experiments were performed the next dayon conscious animals. During the interim, catheter patency wasmaintained by constant infusion of saline (0.2 ml/h). On the day of theexperiment, the animals were intravenously injected with rPH.2 orvehicle control, followed by an infusion of either (1) 5 μg/kg per hourof caerulein for 3.5 hours, or (2) 10 μg/kg per hour of caerulein for 5hours, (Research Plus, Bayonne, N.J.). Immediately after completion ofthe infusion, the animals were anesthetized with pentobarbital sodium,their abdomens were opened, and 5 ml of blood aspirated from theinferior vena cava for subsequent assays. They were then sacrificed byexsanguination. Serum amylase, serum lipase and serum bilirubin weremeasured, and the pancreas was harvested. Pieces of pancreas were eitherfixed in a 4% phosphate buffered formaldehyde solution for histologicalexamination or immediately deep frozen at -80° C. for measurements ofmyeloperoxidase activity. Additional pieces of pancreas were assessedfor pancreatic water content and pancreatic amylase and trypsin asdescribed below. Myeloperoxidase activity, a measure of neutrophilsequestration, was assessed in the pancreas and lung as described below.Pulmonary vascular permeability was also assessed as described below.Statistical analysis of the data was accomplished using unpairedStudent's t-test. The data reported represent means±S.E.M. of at leastthree different experiments. Differences in the results were consideredsignificant when p<0.05.

1. Pancreatic water content

Pancreas pieces were blotted dry and weighed (wet weight), and were thendesiccated for 34 hrs at 120° C. and reweighed (dry weight). Pancreaticwater content was calculated as the difference between wet and dryweight and expressed as a percentage of the pancreatic wet weight. Arise in pancreatic water content was considered to indicate thedevelopment of edema.

2. Serum and Pancreatic Amylase

Amylase activity in serum was measured using 4,6-ethylidene(G₇)-p-nitrophenyl (G₁)-α₁ D-maltoplaside (ET-G₇ PNP) (Sigma ChemicalCo., St. Louis, Mo.) as substrate according to Pierre et al., Clin.Chem., 22:1219 (1976). Amylase activity in pancreatic tissue homogenizedin 10 mM phosphate buffer, pH 7.4, was measured using the same method.

3. Pancreatic Trypsin

Trypsin activity was measured fluorimetrically using Boc-Gin-Ala-Arg-MCAas the substrate. Briefly, 200 μl of the sample and 2.7 ml of 50 mMTris-buffer (pH 8.0) containing 150 mM NaCl, 1 mM CaCl₂ and 0.1% bovineserum albumin were mixed in a cuvette. One hundred μl of substrate wasadded to the sample after 20 seconds of preincubation to start thereaction. The fluorescence reading was taken (excitation 380 nm,emission 440 nm) and expressed as slope. To allow pooling of data fromdifferent experiments trypsin activity in the fractions was expressed aspercent of total trypsin activity.

4. Histology and Morphometry

For light microscopy, complete random cross-sections of the head, bodyand tail of the pancreas were fixed in 10% neutral phosphate-bufferedformalin. Paraffin embedded-5 μm sections were stained withhematoxylin-eosin (H&E) and examined in a blinded fashion by anexperienced morphologist. Acinar cell injury/necrosis was defined aseither (a) the presence of acinar cell ghosts or (b) vacuolization andswelling of acinar cells and destruction of the histo-architecture ofwhole or parts of the acini, both of which had to be associated with aninflammatory reaction. The amount of acinar cell injury/necrosis and thetotal area occupied by acinar tissue were each quantitatedmorphometrically using computerized planimetric image analysis videounit (model CCD-72, Dage-MT1, Michigan city, Ind.) equipped withNIH-1200 image analysis software. Ten randomly chosen microscopic fields(125×) were examined for each tissue sample. The extent of acinar cellinjury/necrosis was expressed as the percent of total acinar tissuewhich was occupied by areas which met the criteria for injury/necrosis.

5. Pancreas and Lung Myleoperoxidase (MPO) Activity Measurement

Neutrophil sequestration in pancreas and lung was evaluated bymeasurement of tissue myeloperoxidase activity. Tissue samples harvestedat the time of sacrifice were stored at -70° C. until the time of assay.Samples (50 mg) were thawed and homogenized in 1 mL of 20 mM phosphatebuffer (pH 7.4) and centrifuged (10,000×g, 10 min 4° C.). The resultingpellet was resuspended in 50 mM phosphate buffer (pH 6.0) containing0.5% hexadecyltrimethylammonium bromide (Sigma, St. Louis, Mo.) andsubjected to four cycles of freezing-thawing. The suspension was thenfurther disrupted by sonication for 40 sec. and centrifuged (10,000×g, 5min. at 4° C.). A reaction mixture consisting of the extracted enzyme,1.6 mM tetramethylbenzidine (Sigma Chemical Co., St. Louis, Mo.), 80 mMsodium phosphate buffer (pH 5.4) and 0.3 mM hydrogen peroxide wasincubated at 37° C. for 110 sec, and the absorbance was measured at 655nm in a CobasBio autoanalyzer. This absorbance was then corrected forthe fraction dry weight of the tissue sample.

6. Measurement of Pulmonary Vascular Permeability

Obstruction of the common biliopancreatic duct also typically results insevere pancreatitis-associated lung injury quantifiable by lung vascularpermeability and histological examination.

Two hours before the animals were killed, an intravenous bolus injectionof 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, SigmaChemical Co., St. Louis, Mo.) was given. Pulmonary microvascularpermeability was evaluated by quantifying the leakage of FITC-albuminfrom the vascular compartment into the bronchoalveolar space. Briefly,just after sacrifice, the right bronchus was blocked using a clamp andthe trachea exposed. Subsequently, the right lung was lavaged by using acannula inserted into the trachea. Three washes of saline (60 ml ravage)were pooled and the FITC fluorescence in serum and lavage was measuredat excitation 494 nm and emission 520 nm. The fluorescence ratio oflavage fluid to blood was calculated and taken as a measure ofmicrovascular permeability in the lung. The lung was also stained withH&E and examined histologically.

7. Effect of Caerulein and rPH.2 administration

Infusion of caerulein alone at 5 μg/kg/h for 3.5 hours resulted in atypical mild secretagogue-induced pancreatitis in the rats, which wascharacterized by hyperamylasemia, pancreatic edema as measured bypancreatic water content, and histological changes including markedacinar cell vacuolization and pancreatic edema. Saline infusion incontrol animals did not result in any of these biochemical orhistological changes. Administration of rPH.2 intravenously at doses of5, 10 or 20 mg/kg 30 min. before the start of caerulein infusion did notsignificantly alter the magnitude of the changes in pancreatic edema(water content) and histology that were induced by infusion of caeruleinalone. Administration of rPH.2 also had no effect on caerulein-inducedactivation of pancreatic trypsinogen or amylase content.

Infusion of a higher dose of caerulein, 10 μg/kg/h for 5 hours, to ratsresulted in a more severe pancreatitis, characterized relative to thecontrols by a more pronounced increase in serum amylase activity andpancreatic edema, a marked increase in pancreatic MPO activity, and asignificant increase in trypsinogen activation and amylase activity inthe pancreas. Pancreatic histology indicated not only pancreatic edemaand acinar cell vacuolization but also some patchy necrosis and a fewinfiltrating cells.

Administration of rPH.2 (5 or 10 mg/kg intravenously) 30 min. before thestart of caerulein (10 μg/kg/h) infusion ameliorated the magnitude ofmany of the pancreatic changes induced by the infusion of caeruleinalone. Results are shown in Table 10 below. rPH.2 treatment at a dose of5 mg/kg resulted in decrease of serum amylase activity (from 10984±1412to 6763±1256). The higher 10 mg/kg dose of rPH.2 did not result infurther improvement of hyperamylasemia. Treatment with either 5 or 10mg/kg rPH.2 also resulted in some decrease in caerulein-induceddevelopment of pancreatic edema as measured by water content (90.61±0.27for caerulein alone vs. 88.21±0.61 for caerulein +5 mg/kg rPH.2). The 5mg/kg dose of rPH.2 provided a significant amelioration of pancreaticMPO activity (2.92+0.32 fold increase over controls for caerulein alonevs. 1.19+0.21 for caerulein with rPH.2, p<0.05). Higher doses of rPH.2did not result in further improvement of MPO activity. Neither dose ofrPH.2 significantly altered the extent of trypsinogen activation or theamylase content in the pancreas. Pancreatic histology indicated someimprovement in microscopic necrosis and infiltration after rPH.2pretreatment.

Pancreatitis associated lung injury has been observed both clinicallyand in several models of pancreatitis. Infusion of caerulein at 5μg/kg/h for 3.5 h, which resulted in a mild form of pancreatitis, didnot result in significant injury to the lungs. However, infusion ofcaerulein at 10 μg/kg/h for 5 hours, which resulted in more severepancreatitis, also resulted in lung injury quantified by increased lungvascular permeability (0.31±0.04 to 0.79±0.09), lung MPO activity(indicating neutrophil sequestration) and neutrophil infiltration onhistological examination.

Administration of rPH.2 at a dose of 5 mg/kg 30 min prior to caeruleininfusion significantly ameliorated the rise in lung MPO activity inducedby the infusion of caerulein alone (3.55±0.93 for caerulein alone vs.1.51±0.26 for caerulein with rPH.2). rPH.2 treatment significantlydecreased the severity of microscopic changes observed in the lungtissue after caerulein infusion. The caerulein-induced increase in lungvascular permeability was reduced by rPH.2 treatment, although notstatistically significant. The higher 10 mg/kg dose of rPH.2 was no moreeffective than the lower dose in decreasing the severity ofcaerulein-induced lung injury.

                  TABLE 10                                                        ______________________________________                                                      Caerulein  CER +     CER +                                      Control       (CER)      5 mg/kg   10 mg/kg                                   (no CER)      10 μg/kg/h                                                                            rPH.2     rPH.2                                      ______________________________________                                        Serum   961 ± 174                                                                            10984 ± 1412                                                                          6763 ± 1256                                                                        8576 ± 1024                           Amylase                                                                       (U/l)                                                                         Pancreas                                                                              72.71 ± 0.64                                                                         90.61 ± 0.27                                                                          88.21 ±                                                                            89.00 ±                               Water                        0.61    0.94                                     Content                                                                       (% wet                                                                        weight)                                                                       Pancreas                                                                              1.0       2.92 ± 0.32                                                                           1.19 ± 0.21                                                                        1.42 ± 0.19                           MPO (fold                                                                     increase                                                                      over control)                                                                 Pancreas                                                                              0.12 ± 0.06                                                                          9.70 ± 2.50                                                                           8.33 ± 1.75                                                                        9.15 ± 1.28                           Trypsin                                                                       Activity                                                                      (1000xslope/                                                                  μg DNA                                                                     Pancreas                                                                              0.28 ± 0.06                                                                          0.42 ± 0.07                                                                           0.45 ± 0.04                                                                        0.46 ± 0.044                          Amylase                                                                       Content                                                                       (U/μg DNA)                                                                 Lung    0.31 ± 0.04                                                                          0.79 ± 0.09                                                                           0.70 ± 0.09                                                                        0.70 ± 0.07                           Vascular                                                                      Permeability                                                                  (Lavage/                                                                      Serum %)                                                                      Lung MPO                                                                              1.0       3.55 × 0.93                                                                        1.51 ± 0.26                                                                        1.64 ± 0.22                           (fold                                                                         increase                                                                      over control)                                                                 ______________________________________                                    

B. Activity in an Opossum Pancreatitis Model

Healthy, randomly trapped American opossums (Didelphis virginiana) ofeither sex (2.0 kg to 4.0 kg) were obtained from Scott-Haas and housedin climate controlled rooms at 23±2° C. with a 12 hour light/dark cycleand fed a standard laboratory chow with water ad libitum. After anovernight fast, the animals were anesthetized with 50 mg/kgsodium-pentobarbital i.p. (Veterinary Laboratories Inc., Lenexa, Kans.).A celiotomy was performed through a midline incision under sterileconditions and the common bile pancreatic duct was ligated in allanimals to induce acute necrotizing pancreatitis. Additionally, thecystic duct was ligated to prevent the gallbladder from serving as abile reservoir. The animals were randomly assigned to either control orexperimental groups. Starting at Day 2 after ligation of the pancreaticduct, the experimental group received 5 mg/kg body weight per day ofrPH.2 (supplied in a 4 mg/ml solution) intravenously via the tail vein,while the control group received an intravenous injection of the samevolume of placebo vehicle only. After 1 and 2 days of treatment (at Day3 and Day 4 after ligation of the pancreatic duct) the animals wereeuthanized by a sodium-pentobarbital overdose. Blood samples were drawnfrom the heart for measurements of serum amylase, serum lipase and serumbilirubin, and the pancreas was harvested. Pieces of pancreas wereeither fixed in a 4% phosphate buffered formaldehyde solution forhistological examination or immediately deep frozen at -80° C. formeasurements of myeloperoxidase activity. Additional pieces of pancreaswere assessed for pancreatic water content and pancreatic amylase asdescribed above in section A of this example. Myeloperoxidase activity,a measure of neutrophil sequestration, was assessed in the pancreas asdescribed above. Pulmonary vascular permeability was also assessed asdescribed above.

The results reported represent mean±standard error of the mean (SEM)values obtained from multiple determinations in 3 or more separateexperiments. The significance of changes was evaluated using Student'st-test when the data consisted of only two groups or by analysis ofvariance (ANOVA) when comparing three or more groups. If ANOVA indicateda significant difference, the data were analyzed using Tukey's method asa post hoc test for the difference between groups. A p-value of <0.05was considered to indicate a significant difference

Results are shown in Table 11. Obstruction of the common biliopancreaticduct resulted in severe necrotizing pancreatitis characterized byhyperamylasemia, hyperlipasemia and extensive necrosis of the pancreas.Furthermore, obstruction of the common biliopancreatic duct wasassociated with an marked increase in serum bilirubin levels.Intravenous administration of rPH.2 (5 mg/kg/day) starting at Day 2after ligation of the pancreatic duct ameliorated the magnitude of manyof the pancreatic changes induced by duct obstruction and placebotreatment alone. One day of rPH.2 treatment reduced serum amylase levelsin comparison to placebo treated animals, although the difference wasnot statistically significant, and two days of rPH.2 treatment (at Day 4after ligation of the pancreatic duct) significantly reduced serumamylase levels compared to placebo. One or two days of rPH.2 treatmentreduced serum lipase levels relative to controls, although thedifference was not statistically significant. Two days of rPH.2treatment reduced pancreatic amylase content relative to controls,although one day of treatment resulted in an increase in pancreaticamylase. Treatment with rPH.2 was not observed to affect serum bilirubinlevels, pancreas myeloperoxidase activity or pancreas water content.

The major characteristic histological changes induced by obstruction ofthe biliopancreatic duct included marked necrosis, infiltration ofinflammatory cells, acinar cell vacuolization, and marked distention ofthe acinar lumina. Morphometrical examination of the pancreas for acinarcell injury showed a major protective effect of rPH.2 on the pancreasafter one and two days of rPH.2 treatment. After one day of rPH.2treatment, the acinar cell injury was reduced to about 23% of totalacinar cell tissue, compared to 48% injury for the placebo-treatedanimals. This reduction of acinar cell injury was even more pronouncedafter two days of treatment, at which time rPH.2 treatment resulted inabout 35% injury of the total acinar cell tissue, compared to about 60%injury for the placebo-treated animals.

Lung vascular permeability, quantified by FITC injection showed a highlysignificant difference after one and two days of rPH.2 treatmentcompared to placebo group. Histological examination of the lung showedsevere lung injury in all placebo-treated animals. Lung injury wascharacterized by an extensive inflammatory response with interstitialand intraalveolar infiltration of mainly macrophages, lymphocytes andneutrophils, and by a patchy but marked interstitial edema andthickening of the alveolar membranes. Administration of rPH.2 resultedin a marked decrease of infiltration of inflammatory cells and areduction of interstitial edema at all times.

In summary, these results showed that administration of rPH.2intravenously at a dose of 5 mg/kg/day beginning at 48 hours afterligation of the pancreatic duct resulted in significant amelioration ofthe increase in blood levels of amylase and lipase and acinar cellinjury as quantitated by morphometric analysis of H&E stained sections,and a significant decrease in the severity of pancreatitis-induced lunginjury. Administration of rPAF-AH product in this clinically relevantmodel of pancreatitis showed beneficial effects in decreasing theseverity of pancreatitis.

                                      TABLE 11                                    __________________________________________________________________________           After 1 day of treatment                                                                     After 2 days of treatment                                      (Sacrifice at Day 3)                                                                         (Sacrifice at Day 4)                                                 rPH.2           rPH.2                                                   Placebo                                                                             5mg/kg   Placebo                                                                              5 mg/kg                                          __________________________________________________________________________    Serum bilirubin                                                                       5.49 ± 0.96                                                                      7.10 ± 0.60                                                                         6.54 ± 0.55                                                                       4.91 ± 0.79                                  (mg/dl)                                                                       Serum amylase                                                                        5618 ± 899                                                                       4288 ± 675                                                                           6538 ± 1355                                                                       3106 ± 467*                                  (U/l)                                                                         Serum lipase                                                                         2226 ± 554                                                                       1241 ± 263                                                                          1424 ± 257                                                                        1023 ± 295                                    (U/l)                                                                         Pancreas Water                                                                       81.10 ± 0.56                                                                     81.52 ± 0.79                                                                        80.05 ± 1.07                                                                      79.32 ± 0.49                                  Content (%)                                                                   Pancreas MPO                                                                         1345 ± 286                                                                       1142 ± 83                                                                           1149 ± 232                                                                        1033 ± 130                                    (OD/fraction                                                                  dry weight)                                                                   Pancreatic                                                                           706 ± 92                                                                         1101 ± 105                                                                          950 ± 85                                                                           712 ± 131                                    Amylase                                                                       U/μg DNA)                                                                  Lung Vascular                                                                         0.76 ± 0.09                                                                      0.21 ± 0.04**                                                                       0.57 ± 0.13                                                                       0.23 ± 0.04*                                 Permeability                                                                  (FITC Lavage/                                                                 Serum %)                                                                      Acinar Cell                                                                          48%   23%      60%    35%                                              Injury (% of                                                                  Total Acinar                                                                  Tissue)                                                                       __________________________________________________________________________     *p = 0.02 vs. placebo                                                         **p < 0.001 vs. placebo                                                  

EXAMPLE 20

A study was conducted to evaluate the effect of a PAF-AH product, rPH.2,on neurotoxicity associated with HIV infection. Human immunodeficiencyvirus type 1 (HIV-1) infection of the central nervous system results inneuronal loss by apoptosis. HIV-1-infected monocytes activated by avariety of antigenic stimuli, including contact with neural cells,secrete high levels of neurotoxic pro-inflammatory cytokines, includingPAF. The effect of rPH.2 on the neurotoxicity of conditioned media fromHIV-infected and activated monocytes was assessed.

Monocytes were infected with HIV and activated as follows. Monocyteswere recovered from peripheral bone marrow cells (PBMC) of HIV- andhepatitis B-seronegative donors after leukopheresis and purified (>98%)by countercurrent centrifugal elutriation as described in Genis et al.,J. Exp. Med., 176:1703-1718 (1992). Cells were cultured as adherentmonolayers (1×10⁴ cells/ml in T-75 culture flasks) in DMEM (Sigma, St.Louis, Mo.) with recombinant human macrophage colony stimulatory factor(MSCF) (Genetics Institute, Inc. Cambridge, Mass.). Under theseconditions, monocytes differentiate into macrophages. After 7-10 days ofculture, macrophages were; exposed to HIV-1_(ADA) (accession numberM60472) at a multiplicity of infection (MOI) of 0.01 infectiousvirions/target cell. Under these conditions, 20-50% of the monocyteswere infected at 7 days after HIV-1 inoculation, as determined byimmunofluorescent and in situ hybridization techniques [Kalter et al.,J. Immunol., 146:298-306 (1991)]. All cultures were refed with freshmedium every 2 to 3 days. Five to seven days after HIV-1 infection andduring the peak of reverse transcriptase activity (10⁷ cpm/ml), assessedaccording to Kalter et al., supra, cultures of HIV-1-infected andparallel cultures of uninfected monocytes were stimulated with LPS (10ng/ml) or vehicle for 30 min. at 37° C., then snap-frozen at -80° C.until used in the neurotoxicity assay.

Human cerebral cortical neuron cell cultures were established asfollows. Human fetal brain tissue was obtained from the telencephalon ofsecond trimester (13-16 weeks gestation) human fetal brain tissueaccording to a modified procedure of Banker and Cowan, Brain Res.,126:397-425 (1977). Briefly, brain tissue was collected, washed in 30 mlof cold Hank's BSS (containing Ca⁺² and Mg⁺² ±25 mM HEPES, and 5×gentamicin), separated from adherent meninges and blood, and cut into 2mm³ pieces. The tissue was forced through a 230 μM Nitex bag and gentlytriturated through a flame-polished Pasteur pipet 10-15 times. Thetissue was centrifuged at 550 rpm, 5 minutes, 4° C., and the pellet wasresuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/liter;L-glutamine, 2 mM; HEPES, 10 mM; Na pyruvate, 1 mM; KCl, 20 mM)containing N1 components (insulin, 5 mg/l; transferrin, 5 mg/l;selenite, 5 μg/l, progesterone 20 nM; putrescine, 100 μM), as well as10% fetal calf serum (FCS), PSN antibiotic mix (penicillin, 50 mg/l;streptomycin, 50 mg/l; neomycin, 100 mg/l), and fungizone (2.5 mg/l).The cell count and viability were determined by diluting Hank's BSS with0.4% trypan blue (1:1 v/v) and counting with a hemocytometer. Cells weretently triturated 5 times with a 10 ml pipet and plated at a density of10⁵ cells/12 mm glass coverslip pre-coated with poly-L-lysine (70K-150KMW, Sigma, St. Louis, Mo.) placed in 24 well culture dishes. One ml ofmedia was pipetted into each culture well. Cells were cultured for 10-28days at 37° C. in a humidified atmosphere of 5% CO₂ /95% air, changingmedia every 3 days. Under these conditions, cultures were >60-70%homogeneous for neurons, with 20-30% astrocytes, <1% microglia and ˜10%macrophage and microglia staining. After 14-28 days of culture, neuronalcultures express sufficient levels of N-methyl-D-aspartate (NMDA) ornon-NMDA receptors to die after excitotoxic doses of NMDA oralpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA).

The neurotoxicity assay was conducted as follows. The test samples,which were (a) conditioned media from LPS-stimulated HIV-1 infectedmonocytes, (b) control media, (c) conditioned media with added rPH.2 at51 μg/ml or (d) conditioned media with added vehicle for rPH.2, wereapplied to the neuronal cell cultures at a 1:10 v/v concentration for 24hours. Neurotoxicity was measured by identifying apoptotic nuclei insitu on neuronal coverslips fixed in 4% paraformaldehyde, employing acommercial kit (Apop Tag; ONCOR, Gaithersburg, Md.) that uses terminaldeoxynucleotidyl transferase (TdT) to bind digoxigenin-dUPT to free3'-OH ends of newly cleaved DNA (TUNEL staining). Digitized images ofTUNEL-stained neurons in ≧15 randomly selected microscopic fields wereanalyzed for number of TUNEL-stained nuclei/number of total neurons per50× field using computerized morphometry (MCID, Imaging Research, St.Catherine, Ontario, Canada). Data were expressed at %neuronal nucleipositive for TUNEL staining±SEM and are shown in FIG. 13. Tests ofstatistical significance between control and experimental treatmentswere determined by ANOVA or paired t-tests, with significance at p≦0.05.Quantitation of these cultures confirmed that conditioned media fromHIV-infected and activated monocytes induced neuronal cell death innearly 25% of the total population of cerebral cortical neurons, andrPH.2 was able to reduce this toxicity to less than 5% of the totalneurons. The rPH.2 by itself was not neurotoxic, since 50 μg/ml rPH.2had no effect on neuronal cell death relative to cultures treated withcontrol media. These results clearly indicate that a major component ofthe neurotoxicity induced by application of conditioned media fromactivated HIV-1 infected monocytes must be due to PAF, since neurotoxitycan be almost completely abrogated by co-incubation with PAF-AH product,the enzyme responsible for metabolism of PAF in the central nervoussystem. These findings suggest potential therapeutic interventions inthe treatment of the CNS neurologic disease associated with HIV-1infection.

EXAMPLE 21

Nearly four percent of the Japanese population has low or undetectablelevels of PAF-AH activity in their plasma. This deficiency has beencorrelated with severe respiratory symptoms in asthmatic children [Miwaet al., J. Clin. Invest,. 82: 1983-1991 (1988)] who appear to haveinherited the deficiency in an autosomal recessive manner.

To determine if the deficiency arises from an inactive but presentenzyme or from an inability to synthesize PAF-AH, plasma from multiplepatients deficient in PAF-AH activity was assayed both for PAF-AHactivity (by the method described in Example 10 for transfectants) andfor the presence of PAF-AH using the monoclonal antibodies 90G11D and90F2D (Example 13) in a sandwich ELISA as follows. Immulon 4 flat bottomplates (Dynatech, Chantilly, Va.) were coated with 100 ng/well ofmonoclonal antibody 90G11D and stored overnight. The plates were blockedfor 1 hour at room temperature with 0.5% fish skin gelatin (Sigma)diluted in CMF-PBS and then washed three times. Patient plasma wasdiluted in PBS containing 15 mM CHAPS and added to each well of theplates (50 μl/well). The plates were incubated for 1 hour at roomtemperature and washed four times. Fifty μl of 5 μg/ml monoclonalantibody 90F2D, which was biotinylated by standard methods and dilutedin PBST, was added to each well, and the plates were incubated for 1hour at room temperature and then washed three times. Fifty μl ofExtraAvidin (Sigma) diluted 1/1000 in CMF-PBST was subsequently added toeach well and plates were incubated for 1 hour at room temperaturebefore development.

A direct correlation between PAF-AH activity and enzyme levels wasobserved. An absence of activity in a patient's serum was reflected byan absence of detectable enzyme. Similarly, plasma samples with half thenormal activity contained half the normal levels of PAF-AH. Theseobservations suggested that the deficiency of PAF-AH activity was due toan inability to synthesize the enzyme or due to an inactive enzyme whichthe monoclonal antibodies did not recognize.

Further experiments revealed that the deficiency was due to a geneticlesion in the human plasma PAF-AH gene. Genomic DNA from PAF-AHdeficient individuals was isolated and used as template for PCRreactions with PAF-AH gene specific primers. Each of the coding sequenceexons were initially amplified and sequenced from one individual. Asingle nucleotide change within exon 9 was observed (a G to T atposition 996 of SEQ ID NO: 7). The nucleotide change results in an aminoacid substitution of a phenylalanine for a valine at position 279 of thePAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from anadditional eleven PAF-AH deficient individuals who were found to havethe same point mutation.

To test whether this mutation crippled the enzyme, an E. coli expressionconstruct containing the mutation was generated by methods similar tothat described in Example 10. When introduced into E. coli, theexpression construct generated no PAF-AH activity while a controlconstruct lacking the mutation was fully active. This amino acidsubstitution presumably results in a structural modification whichcauses the observed deficiency of activity and lack of immunoreactivitywith the PAF-AH antibodies of the invention.

PAF-AH specific antibodies of the invention may thus be used indiagnostic methods to detect abnormal levels of PAF-AH in serum (normallevels are about 1 to 5 U/ml) and to follow the progression of treatmentof pathological conditions with PAF-AH. Moreover, identification of agenetic lesion in the PAF-AH gene allows for genetic screening for thePAF-AH deficiency exhibited by the Japanese patients. The mutationcauses the gain of a restriction endonuclease site (Mae II) and thusallows for the simple method of Restriction Fragment Length Polymorphism(RFLP) analysis to differentiate between active and mutant alleles. SeeLewin, pp. 136-141 in Genes V, Oxford University Press, New York, N.Y.(1994).

Screening of genomic DNA from twelve PAF-AH deficient patients wascarried out by digestion of the DNA with MaeII, Southern blotting, andhybridization with an exon 9 probe (nucleotides 1-396 of SEQ ID NO: 17).All patients were found to have RFLPs consistent with the mutant allele.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the appended claims should be placed on the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 30                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Al - #a Leu Val Leu Ile Ala         #                15                                                           - Phe                                                                         - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gl - #y Gln Thr Lys Ile Pro         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Lys Pro Leu Val Val Phe Val Leu Gly Gl - #y                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                     #21, 27)  (B) LOCATION: group(13,                                             #/note= "The nucleotide at each of                                                           these pos - #itions is an inosine."                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #          32      YTTG NGTYTGNCCR AA                                         - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #           30     GTGG AACTCGCTGG                                            - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #          32      CAGC AGCCATCAGT AC                                         - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1520 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 162..1484                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GGGTTTGGAG CG - #CTTGGGTC         60                                                                          - GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CCGCGAGCAG CT - #CCGCGCCG        120                                                                          #CCA CCC        173CTGA AACTGCTGCT CAGCTCCCAA G ATG GTG                       #          Met Val Pro Pro                                                    #            1                                                                - AAA TTG CAT GTG CTT TTC TGC CTC TGC GGC TG - #C CTG GCT GTG GTT TAT          221                                                                          Lys Leu His Val Leu Phe Cys Leu Cys Gly Cy - #s Leu Ala Val Val Tyr           #  20                                                                         - CCT TTT GAC TGG CAA TAC ATA AAT CCT GTT GC - #C CAT ATG AAA TCA TCA          269                                                                          Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Al - #a His Met Lys Ser Ser           #                 35                                                          - GCA TGG GTC AAC AAA ATA CAA GTA CTG ATG GC - #T GCT GCA AGC TTT GGC          317                                                                          Ala Trp Val Asn Lys Ile Gln Val Leu Met Al - #a Ala Ala Ser Phe Gly           #             50                                                              - CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TA - #T TCC GTT GGT TGT ACA          365                                                                          Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Ty - #r Ser Val Gly Cys Thr           #         65                                                                  - GAC TTA ATG TTT GAT CAC ACT AAT AAG GGC AC - #C TTC TTG CGT TTA TAT          413                                                                          Asp Leu Met Phe Asp His Thr Asn Lys Gly Th - #r Phe Leu Arg Leu Tyr           #     80                                                                      - TAT CCA TCC CAA GAT AAT GAT CGC CTT GAC AC - #C CTT TGG ATC CCA AAT          461                                                                          Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Th - #r Leu Trp Ile Pro Asn           #100                                                                          - AAA GAA TAT TTT TGG GGT CTT AGC AAA TTT CT - #T GGA ACA CAC TGG CTT          509                                                                          Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Le - #u Gly Thr His Trp Leu           #               115                                                           - ATG GGC AAC ATT TTG AGG TTA CTC TTT GGT TC - #A ATG ACA ACT CCT GCA          557                                                                          Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Se - #r Met Thr Thr Pro Ala           #           130                                                               - AAC TGG AAT TCC CCT CTG AGG CCT GGT GAA AA - #A TAT CCA CTT GTT GTT          605                                                                          Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Ly - #s Tyr Pro Leu Val Val           #       145                                                                   - TTT TCT CAT GGT CTT GGG GCA TTC AGG ACA CT - #T TAT TCT GCT ATT GGC          653                                                                          Phe Ser His Gly Leu Gly Ala Phe Arg Thr Le - #u Tyr Ser Ala Ile Gly           #   160                                                                       - ATT GAC CTG GCA TCT CAT GGG TTT ATA GTT GC - #T GCT GTA GAA CAC AGA          701                                                                          Ile Asp Leu Ala Ser His Gly Phe Ile Val Al - #a Ala Val Glu His Arg           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - GAT AGA TCT GCA TCT GCA ACT TAC TAT TTC AA - #G GAC CAA TCT GCT GCA          749                                                                          Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Ly - #s Asp Gln Ser Ala Ala           #               195                                                           - GAA ATA GGG GAC AAG TCT TGG CTC TAC CTT AG - #A ACC CTG AAA CAA GAG          797                                                                          Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Ar - #g Thr Leu Lys Gln Glu           #           210                                                               - GAG GAG ACA CAT ATA CGA AAT GAG CAG GTA CG - #G CAA AGA GCA AAA GAA          845                                                                          Glu Glu Thr His Ile Arg Asn Glu Gln Val Ar - #g Gln Arg Ala Lys Glu           #       225                                                                   - TGT TCC CAA GCT CTC AGT CTG ATT CTT GAC AT - #T GAT CAT GGA AAG CCA          893                                                                          Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Il - #e Asp His Gly Lys Pro           #   240                                                                       - GTG AAG AAT GCA TTA GAT TTA AAG TTT GAT AT - #G GAA CAA CTG AAG GAC          941                                                                          Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Me - #t Glu Gln Leu Lys Asp           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - TCT ATT GAT AGG GAA AAA ATA GCA GTA ATT GG - #A CAT TCT TTT GGT GGA          989                                                                          Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gl - #y His Ser Phe Gly Gly           #               275                                                           - GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CA - #G AGA TTC AGA TGT GGT         1037                                                                          Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gl - #n Arg Phe Arg Cys Gly           #           290                                                               - ATT GCC CTG GAT GCA TGG ATG TTT CCA CTG GG - #T GAT GAA GTA TAT TCC         1085                                                                          Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gl - #y Asp Glu Val Tyr Ser           #       305                                                                   - AGA ATT CCT CAG CCC CTC TTT TTT ATC AAC TC - #T GAA TAT TTC CAA TAT         1133                                                                          Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Se - #r Glu Tyr Phe Gln Tyr           #   320                                                                       - CCT GCT AAT ATC ATA AAA ATG AAA AAA TGC TA - #C TCA CCT GAT AAA GAA         1181                                                                          Pro Ala Asn Ile Ile Lys Met Lys Lys Cys Ty - #r Ser Pro Asp Lys Glu           325                 3 - #30                 3 - #35                 3 -       #40                                                                           - AGA AAG ATG ATT ACA ATC AGG GGT TCA GTC CA - #C CAG AAT TTT GCT GAC         1229                                                                          Arg Lys Met Ile Thr Ile Arg Gly Ser Val Hi - #s Gln Asn Phe Ala Asp           #               355                                                           - TTC ACT TTT GCA ACT GGC AAA ATA ATT GGA CA - #C ATG CTC AAA TTA AAG         1277                                                                          Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly Hi - #s Met Leu Lys Leu Lys           #           370                                                               - GGA GAC ATA GAT TCA AAT GTA GCT ATT GAT CT - #T AGC AAC AAA GCT TCA         1325                                                                          Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Le - #u Ser Asn Lys Ala Ser           #       385                                                                   - TTA GCA TTC TTA CAA AAG CAT TTA GGA CTT CA - #T AAA GAT TTT GAT CAG         1373                                                                          Leu Ala Phe Leu Gln Lys His Leu Gly Leu Hi - #s Lys Asp Phe Asp Gln           #   400                                                                       - TGG GAC TGC TTG ATT GAA GGA GAT GAT GAG AA - #T CTT ATT CCA GGG ACC         1421                                                                          Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu As - #n Leu Ile Pro Gly Thr           405                 4 - #10                 4 - #15                 4 -       #20                                                                           - AAC ATT AAC ACA ACC AAT CAA CAC ATC ATG TT - #A CAG AAC TCT TCA GGA         1469                                                                          Asn Ile Asn Thr Thr Asn Gln His Ile Met Le - #u Gln Asn Ser Ser Gly           #               435                                                           - ATA GAG AAA TAC AAT TAGGATTAAA ATAGGTTTTT TAAAAAAAA - #A AAAAAA             1520                                                                          Ile Glu Lys Tyr Asn                                                                       440                                                               - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 441 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Val Pro Pro Lys Leu His Val Leu Phe Cy - #s Leu Cys Gly Cys Leu         #                 15                                                          - Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Il - #e Asn Pro Val Ala His         #             30                                                              - Met Lys Ser Ser Ala Trp Val Asn Lys Ile Gl - #n Val Leu Met Ala Ala         #         45                                                                  - Ala Ser Phe Gly Gln Thr Lys Ile Pro Arg Gl - #y Asn Gly Pro Tyr Ser         #     60                                                                      - Val Gly Cys Thr Asp Leu Met Phe Asp His Th - #r Asn Lys Gly Thr Phe         # 80                                                                          - Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asn As - #p Arg Leu Asp Thr Leu         #                 95                                                          - Trp Ile Pro Asn Lys Glu Tyr Phe Trp Gly Le - #u Ser Lys Phe Leu Gly         #           110                                                               - Thr His Trp Leu Met Gly Asn Ile Leu Arg Le - #u Leu Phe Gly Ser Met         #       125                                                                   - Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Ar - #g Pro Gly Glu Lys Tyr         #   140                                                                       - Pro Leu Val Val Phe Ser His Gly Leu Gly Al - #a Phe Arg Thr Leu Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gl - #y Phe Ile Val Ala Ala         #               175                                                           - Val Glu His Arg Asp Arg Ser Ala Ser Ala Th - #r Tyr Tyr Phe Lys Asp         #           190                                                               - Gln Ser Ala Ala Glu Ile Gly Asp Lys Ser Tr - #p Leu Tyr Leu Arg Thr         #       205                                                                   - Leu Lys Gln Glu Glu Glu Thr His Ile Arg As - #n Glu Gln Val Arg Gln         #   220                                                                       - Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Le - #u Ile Leu Asp Ile Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - His Gly Lys Pro Val Lys Asn Ala Leu Asp Le - #u Lys Phe Asp Met Glu         #               255                                                           - Gln Leu Lys Asp Ser Ile Asp Arg Glu Lys Il - #e Ala Val Ile Gly His         #           270                                                               - Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Le - #u Ser Glu Asp Gln Arg         #       285                                                                   - Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Me - #t Phe Pro Leu Gly Asp         #   300                                                                       - Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Ph - #e Phe Ile Asn Ser Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Phe Gln Tyr Pro Ala Asn Ile Ile Lys Me - #t Lys Lys Cys Tyr Ser         #               335                                                           - Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Ar - #g Gly Ser Val His Gln         #           350                                                               - Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Ly - #s Ile Ile Gly His Met         #       365                                                                   - Leu Lys Leu Lys Gly Asp Ile Asp Ser Asn Va - #l Ala Ile Asp Leu Ser         #   380                                                                       - Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys Hi - #s Leu Gly Leu His Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Phe Asp Gln Trp Asp Cys Leu Ile Glu Gl - #y Asp Asp Glu Asn Leu         #               415                                                           - Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn Gl - #n His Ile Met Leu Gln         #           430                                                               - Asn Ser Ser Gly Ile Glu Lys Tyr Asn                                         #       440                                                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1123 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: Not Deter - #mined                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - AAATATAAAT TTTAATAACA CCACACATAA ATTTCAAACT ACTTTCCCTA AG - #TTTCTAGC         60                                                                          - TGAAGTTTTA AATGAGTGTG TTTTTAATTT ATTAGAAAGT GGATTGAAGA GA - #AAACATTG        120                                                                          - GAAGATGAAG GAAGGCGTTT CAGTTAAACC CCAAATAACT CTGTGTTACA CT - #GAGCTATG        180                                                                          - AAACGGCTCC TTCTAGCTCC ATTTCTCCTC AGACCTAAGT GCTATTCCTG AT - #TGTCCTTC        240                                                                          - ATTGTCATTT CCAGGGAGAA ATGACACCAG CACAGTGGCA GGCCTTCCAA TC - #TGGAGCAC        300                                                                          - GGTCCACACA ACTTCCGAAT TGGTGTTCAG TGTAAAGTGT ATCGGAGTGC GG - #AAAATGCG        360                                                                          - CAGGGCATTG CCAACTATAG ATGCTCGGAG TAATTCAGTG TATTCAGAGA AC - #ACGGTGAA        420                                                                          - ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA ATTCAGCAGG GAGTAAATCT GA - #TCGGCATC        480                                                                          - AGGTCTGCGG AAAGGAGCTG GTGAGCACGA CACCACCAGG CATTGCCTGG CT - #CTCTCCGC        540                                                                          - GGCGGGCTAA GTTAACCTCG GGTCCAGGTG CGGGCCATGG TCTTGGGGAG GG - #TGCTGGGT        600                                                                          - GCGCTCGAGC AGGCTACGTC GGGAGCCGCC GCTGCTAGTG AGAGCCGGGC CA - #CACACGCT        660                                                                          - CCTCCCCGGT ACCTCCTCCA GCATCACCAG GGGAGGAGAG GGTCGGGCAC AA - #GGCGCGCT        720                                                                          - AGGCGGACCC AGACACAGCC GCGCGCAGCC CACCCGCCCG CCGCCTGCCA GA - #GCTGCTCG        780                                                                          - GCCCGCAGCC AGGGGGACAG CGGCTGGTCG GAGGCTCGCA GTGCTGTCGG CG - #AGAAGCAG        840                                                                          - TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT GCGCGGTGGA ACCCCCCAGG GA - #CCCCAGTT        900                                                                          - CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT GAGGAGGGGC CCCGGGGGCG AG - #GCGGGAGT        960                                                                          - GGGAGGAAGG GCACGGTCGC CGCGCTGGAG GTCGGGACCC CGGAGCGGCG AC - #CGGCCGGG       1020                                                                          - GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT GGCCGGTCCT GGGCTCACAG TC - #CCTGCAGC       1080                                                                          #                 112 - #3CTTCGGGA GAGGAGAGAT GAC                             - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 417 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 145..287                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTA TTTTTTCCAA GT - #GTTACTAG         60                                                                          - TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA TA - #CTTCTTTT        120                                                                          - TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG CT - #CCCAAGAT        180                                                                          - GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGC TGCCTGGCTG TG - #GTTTATCC        240                                                                          - TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA AG - #AGGTGTAT        300                                                                          - TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CA - #AGAAGTTG        360                                                                          - CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TA - #TTCTC           417                                                                          - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 498 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 251..372                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TC - #ATCTCTTT         60                                                                          - ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TG - #CATATCTT        120                                                                          - ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GA - #TTCCGTTG        180                                                                          - GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AA - #TATTTACC        240                                                                          - TGTTTTTCAG CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC GT - #TTGGCCAA        300                                                                          - ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AA - #TGTTTGAT        360                                                                          - CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA AT - #ATTGTCTG        420                                                                          - TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GA - #ATGACTCA        480                                                                          # 498              GT                                                         - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 130..274                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TA - #ATTAATAT         60                                                                          - CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TT - #AATTTCTT        120                                                                          - TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TC - #ACCTTGAC        180                                                                          - ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TG - #GAACACAC        240                                                                          - TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TC - #CTTCTTTG        300                                                                          - TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TT - #AAGACCAA        360                                                                          - AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TT - #CTTTAAAG        420                                                                          #     433                                                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 486 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 164..257                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AA - #AAAAGTAG         60                                                                          - ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TC - #ACTAGCAA        120                                                                          - CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GA - #CAACTCCT        180                                                                          - GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAATATC CACTTGTTGT TT - #TTTCTCAT        240                                                                          - GGTCTTGGGG CATTCAGGTA ATGTTTGAGA GGTTGAACAA TTTTGGCTTC CA - #GGAATAAA        300                                                                          - TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AG - #GCCCTTGT        360                                                                          - CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CC - #TAGACCTG        420                                                                          - TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CA - #GCAGAGAA        480                                                                          #          486                                                                - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 363 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 113..181                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AG - #TGACTCAG         60                                                                          - CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AG - #GACACTTT        120                                                                          - ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GT - #AGAACACA        180                                                                          - GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AA - #TGCTCATA        240                                                                          - ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TC - #AAGTATGG        300                                                                          - TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AA - #AAGCACCT        360                                                                          #            363                                                              - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 441 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 68..191                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TT - #TTGCTTGT         60                                                                          - ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GC - #TGCAGAAA        120                                                                          - TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG AC - #ACATATAC        180                                                                          - GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AG - #CATGTACA        240                                                                          - AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AG - #CTCTCCTA        300                                                                          - TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CC - #TTAATGGA        360                                                                          - ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG AC - #AGTGAATA        420                                                                          #                 441GG G                                                     - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 577 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 245..358                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AG - #GATTTGGA         60                                                                          - CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AG - #TAATAGTC        120                                                                          - ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GC - #TTCTACCT        180                                                                          - AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AA - #TAATTATA        240                                                                          - TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TT - #GACATTGA        300                                                                          - TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTTT GATATGGAAC AA - #CTGAAGGT        360                                                                          - AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TG - #TCATTTAA        420                                                                          - TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CC - #TCCCCAAT        480                                                                          - GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AG - #AATGCATT        540                                                                          #     577          GAAT GCAAAGTTAG TCAGTTT                                    - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 396 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 108..199                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TC - #ATTTCTTC         60                                                                          - TAACACCACG AAATAGCTAT AAATTTATAT CATGCTTTTT CAAATAGGAC TC - #TATTGATA        120                                                                          - GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT CA - #GACTCTTA        180                                                                          - GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT AG - #TAAATTAT        240                                                                          - TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA GG - #AAGGGGAT        300                                                                          - CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT AC - #ATTTTCCT        360                                                                          #      396         TCTC CTGCTATTAA AATCTC                                     - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 519 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 181..351                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TA - #TTCCAATC         60                                                                          - AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT AT - #TAGGATGT        120                                                                          - CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA AT - #ATCTACAG        180                                                                          - ATGTGGTATT GCCCTGGATG CATGGATGTT TCCACTGGGT GATGAAGTAT AT - #TCCAGAAT        240                                                                          - TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA AT - #ATCATAAA        300                                                                          - AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GG - #TAAGTATT        360                                                                          - AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA AT - #CGATATCA        420                                                                          - TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTCAATAA GG - #AGAGAATT        480                                                                          #   519            TAAG TACATTTAGT CTACTCTTT                                  - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 569 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 156..304                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - TGAAACACAT CTAAGTAGAT CAAATTACAA GTTTTATTTC TTCTTTGGTT TT - #CAGTAAAC         60                                                                          - AGACCAACAA GACCAGTACC TTTCCTTACA CTCTAACTAA AAAAATAATA AT - #TTTATCAA        120                                                                          - ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA GA - #ATTTTGCT        180                                                                          - GACTTCACTT TTGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GG - #GAGACATA        240                                                                          - GATTCAAATG TAGCTATTGA TCTTAGCAAC AAAGCTTCAT TAGCATTCTT AC - #AAAAGCAT        300                                                                          - TTAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG GA - #CAAAGCTG        360                                                                          - TCTATCTTAA TACAGCTTTA GTACTATTTA AACTATTTCC AGTTGGTTTA CA - #ATGGAACA        420                                                                          - AAGCAGTATA TCAATTTGAA AACAGAAATT TGAGAAAGTC AATTTTGCTG CT - #TTACATCT        480                                                                          - CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA GC - #TAGAGTGA        540                                                                          #           569    GAAC GACGGTGCT                                             - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 469 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 137..253                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TA - #AATCTTTA         60                                                                          - TTGCCACCTA GGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATAT GT - #TGATTAAC        120                                                                          - ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GA - #TTGAAGGA        180                                                                          - GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CA - #TCATGTTA        240                                                                          - CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TA - #AAAGTCTT        300                                                                          - GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AG - #AGAGAGAG        360                                                                          - AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TA - #AAATGTAG        420                                                                          #              469TGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT                       - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1494 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 117..1436                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGCATTGCTG CGCTCAGGGT TC - #TGGGTATC         60                                                                          - CGGGAGTCAG TGCAGTGACC AGAACATCAA ACTGAAGCCA CTGCTCAGCT CC - #TAAG            116                                                                          - ATG GTA CCA CTC AAA CTG CAG GCG CTT TTC TG - #C CTC CTC TGC TGC CTC          164                                                                          Met Val Pro Leu Lys Leu Gln Ala Leu Phe Cy - #s Leu Leu Cys Cys Leu           #                 15                                                          - CCA TGG GTC CAT CCT TTT CAC TGG CAA GAC AC - #A TCT TCT TTT GAC TTC          212                                                                          Pro Trp Val His Pro Phe His Trp Gln Asp Th - #r Ser Ser Phe Asp Phe           #             30                                                              - AGG CCG TCA GTA ATG TTT CAC AAG CTC CAA TC - #G GTG ATG TCT GCT GCC          260                                                                          Arg Pro Ser Val Met Phe His Lys Leu Gln Se - #r Val Met Ser Ala Ala           #         45                                                                  - GGC TCT GGC CAT AGT AAA ATC CCC AAA GGA AA - #T GGA TCG TAC CCC GTC          308                                                                          Gly Ser Gly His Ser Lys Ile Pro Lys Gly As - #n Gly Ser Tyr Pro Val           #     60                                                                      - GGT TGT ACA GAT CTG ATG TTC GGT TAT GGG AA - #T GAG AGC GTC TTC GTG          356                                                                          Gly Cys Thr Asp Leu Met Phe Gly Tyr Gly As - #n Glu Ser Val Phe Val           # 80                                                                          - CGT TTG TAC TAC CCA GCT CAA GAT CAA GGT CG - #C CTC GAC ACT GTT TGG          404                                                                          Arg Leu Tyr Tyr Pro Ala Gln Asp Gln Gly Ar - #g Leu Asp Thr Val Trp           #                 95                                                          - ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AG - #T ATA TTT CTT GGA ACA          452                                                                          Ile Pro Asn Lys Glu Tyr Phe Leu Gly Leu Se - #r Ile Phe Leu Gly Thr           #           110                                                               - CCC AGT ATT GTA GGC AAT ATT TTA CAC CTC TT - #A TAT GGT TCT CTG ACA          500                                                                          Pro Ser Ile Val Gly Asn Ile Leu His Leu Le - #u Tyr Gly Ser Leu Thr           #       125                                                                   - ACT CCT GCA AGC TGG AAT TCT CCT TTA AGG AC - #T GGA GAA AAA TAC CCG          548                                                                          Thr Pro Ala Ser Trp Asn Ser Pro Leu Arg Th - #r Gly Glu Lys Tyr Pro           #   140                                                                       - CTC ATT GTC TTT TCT CAT GGT CTC GGA GCC TT - #C AGG ACG ATT TAT TCT          596                                                                          Leu Ile Val Phe Ser His Gly Leu Gly Ala Ph - #e Arg Thr Ile Tyr Ser           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GCT ATT GGC ATT GGC TTG GCA TCT AAT GGG TT - #T ATA GTG GCC ACT GTC          644                                                                          Ala Ile Gly Ile Gly Leu Ala Ser Asn Gly Ph - #e Ile Val Ala Thr Val           #               175                                                           - GAA CAC AGA GAC AGA TCT GCA TCG GCA ACT TA - #C TTT TTT GAA GAC CAG          692                                                                          Glu His Arg Asp Arg Ser Ala Ser Ala Thr Ty - #r Phe Phe Glu Asp Gln           #           190                                                               - GTG GCT GCA AAA GTG GAA AAC AGG TCT TGG CT - #T TAC CTG AGA AAA GTA          740                                                                          Val Ala Ala Lys Val Glu Asn Arg Ser Trp Le - #u Tyr Leu Arg Lys Val           #       205                                                                   - AAA CAA GAG GAG TCG GAA AGT GTC CGG AAA GA - #A CAG GTT CAG CAA AGA          788                                                                          Lys Gln Glu Glu Ser Glu Ser Val Arg Lys Gl - #u Gln Val Gln Gln Arg           #   220                                                                       - GCA ATA GAA TGT TCC CGG GCT CTC AGT GCG AT - #T CTT GAC ATT GAA CAT          836                                                                          Ala Ile Glu Cys Ser Arg Ala Leu Ser Ala Il - #e Leu Asp Ile Glu His           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GGA GAC CCA AAA GAG AAT GTA CTA GGT TCA GC - #T TTT GAC ATG AAA CAG          884                                                                          Gly Asp Pro Lys Glu Asn Val Leu Gly Ser Al - #a Phe Asp Met Lys Gln           #               255                                                           - CTG AAG GAT GCT ATT GAT GAG ACT AAA ATA GC - #T TTG ATG GGA CAT TCT          932                                                                          Leu Lys Asp Ala Ile Asp Glu Thr Lys Ile Al - #a Leu Met Gly His Ser           #           270                                                               - TTT GGA GGA GCA ACA GTT CTT CAA GCC CTT AG - #T GAG GAC CAG AGA TTC          980                                                                          Phe Gly Gly Ala Thr Val Leu Gln Ala Leu Se - #r Glu Asp Gln Arg Phe           #       285                                                                   - AGA TGT GGA GTT GCT CTT GAT CCA TGG ATG TA - #T CCG GTG AAC GAA GAG         1028                                                                          Arg Cys Gly Val Ala Leu Asp Pro Trp Met Ty - #r Pro Val Asn Glu Glu           #   300                                                                       - CTG TAC TCC AGA ACC CTC CAG CCT CTC CTC TT - #T ATC AAC TCT GCC AAA         1076                                                                          Leu Tyr Ser Arg Thr Leu Gln Pro Leu Leu Ph - #e Ile Asn Ser Ala Lys           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - TTC CAG ACT CCA AAG GAC ATC GCA AAA ATG AA - #A AAG TTC TAC CAG CCT         1124                                                                          Phe Gln Thr Pro Lys Asp Ile Ala Lys Met Ly - #s Lys Phe Tyr Gln Pro           #               335                                                           - GAC AAG GAA AGG AAA AAT GAT TAC AAT CAA GG - #G CTC AGG CAC CAG AAC         1172                                                                          Asp Lys Glu Arg Lys Asn Asp Tyr Asn Gln Gl - #y Leu Arg His Gln Asn           #           350                                                               - TTT GAC GAC TTT ACT TTT GTA ACT GGC AAA AT - #A ATT GGA AAC AAG CTG         1220                                                                          Phe Asp Asp Phe Thr Phe Val Thr Gly Lys Il - #e Ile Gly Asn Lys Leu           #       365                                                                   - ACA CTG AAA GGA GAA ATC GAT TCC AGA GTA GC - #C ATC GAC CTC ACC AAC         1268                                                                          Thr Leu Lys Gly Glu Ile Asp Ser Arg Val Al - #a Ile Asp Leu Thr Asn           #   380                                                                       - AAA GCT TCG ATG GCT TTC TTA CAA AAG CAT TT - #A GGG CTT CAG AAA GAC         1316                                                                          Lys Ala Ser Met Ala Phe Leu Gln Lys His Le - #u Gly Leu Gln Lys Asp           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - TTT GAT CAG TGG GAC CCT CTG GTG GAA GGA GA - #T GAT GAG AAC CTG ATT         1364                                                                          Phe Asp Gln Trp Asp Pro Leu Val Glu Gly As - #p Asp Glu Asn Leu Ile           #               415                                                           - CCT GGG TCA CCC TTT GAC GCA GTC ACC CAG GC - #C CCG GCT CAG CAA CAC         1412                                                                          Pro Gly Ser Pro Phe Asp Ala Val Thr Gln Al - #a Pro Ala Gln Gln His           #           430                                                               - TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TG - #CTTGTTAC ACAGTTGCCT        1466                                                                          Ser Pro Gly Ser Gln Thr Gln Asn                                               #       440                                                                   #           1494   CATG AGAGAGAG                                              - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2191 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 92..1423                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CG - #CCCGGAAG         60                                                                          #AAA CTG CAT       112T CAGCTTCCAA G ATG TTG CCA CCC                          #                 Met - # Leu Pro Pro Lys Leu His                             # 1               5                                                           - GCG CTT TTC TGC CTC TGC AGC TGC CTC ACA CT - #G GTT CAT CCT ATT GAC          160                                                                          Ala Leu Phe Cys Leu Cys Ser Cys Leu Thr Le - #u Val His Pro Ile Asp           #         20                                                                  - TGG CAA GAC CTA AAT CCT GTT GCC CAT ATT AG - #A TCA TCA GCA TGG GCC          208                                                                          Trp Gln Asp Leu Asn Pro Val Ala His Ile Ar - #g Ser Ser Ala Trp Ala           #     35                                                                      - AAT AAA ATA CAA GCT CTG ATG GCT GCT GCA AG - #T ATT AGG CAA AGT AGA          256                                                                          Asn Lys Ile Gln Ala Leu Met Ala Ala Ala Se - #r Ile Arg Gln Ser Arg           # 55                                                                          - ATT CCC AAA GGA AAT GGA TCT TAT TCT GTC GG - #T TGT ACA GAT TTG ATG          304                                                                          Ile Pro Lys Gly Asn Gly Ser Tyr Ser Val Gl - #y Cys Thr Asp Leu Met           #                 70                                                          - TTT GAT TAT ACT AAT AAG GGC ACC TTT TTG CG - #T TTG TAT TAT CCA TCG          352                                                                          Phe Asp Tyr Thr Asn Lys Gly Thr Phe Leu Ar - #g Leu Tyr Tyr Pro Ser           #             85                                                              - CAA GAG GAT GAC CAC TCT GAC ACG CTT TGG AT - #C CCA AAC AAA GAA TAT          400                                                                          Gln Glu Asp Asp His Ser Asp Thr Leu Trp Il - #e Pro Asn Lys Glu Tyr           #        100                                                                  - TTT TTT GGT CTT AGT AAA TAT CTT GGA ACA CC - #C TGG CTT ATG GGC AAA          448                                                                          Phe Phe Gly Leu Ser Lys Tyr Leu Gly Thr Pr - #o Trp Leu Met Gly Lys           #   115                                                                       - ATA TTG AGC TTC TTT TTT GGT TCA GTG ACA AC - #T CCT GCG AAC TGG AAT          496                                                                          Ile Leu Ser Phe Phe Phe Gly Ser Val Thr Th - #r Pro Ala Asn Trp Asn           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - TCC CCT CTG AGG ACT GGT GAA AAA TAT CCA CT - #G ATT GTT TTT TCT CAT          544                                                                          Ser Pro Leu Arg Thr Gly Glu Lys Tyr Pro Le - #u Ile Val Phe Ser His           #               150                                                           - GGT CTT GGA GCA TTC CGG ACA ATT TAT TCT GC - #T ATT GGC ATT GAT CTA          592                                                                          Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser Al - #a Ile Gly Ile Asp Leu           #           165                                                               - GCA TCA CAT GGG TTC ATC GTT GCT GCT ATA GA - #A CAC AGA GAT GGA TCC          640                                                                          Ala Ser His Gly Phe Ile Val Ala Ala Ile Gl - #u His Arg Asp Gly Ser           #       180                                                                   - GCC TCT GCG ACT TAC TAT TTC AAG GAC CAG TC - #T GCT GCA GAA ATA GGG          688                                                                          Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gln Se - #r Ala Ala Glu Ile Gly           #   195                                                                       - AAC AAA TCT TGG TCT TAT CTT CAA GAA CTA AA - #A CCA GGG GAT GAG GAG          736                                                                          Asn Lys Ser Trp Ser Tyr Leu Gln Glu Leu Ly - #s Pro Gly Asp Glu Glu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - ATA CAT GTT CGA AAT GAG CAG GTA CAG AAA AG - #G GCA AAG GAG TGC TCC          784                                                                          Ile His Val Arg Asn Glu Gln Val Gln Lys Ar - #g Ala Lys Glu Cys Ser           #               230                                                           - CAA GCT CTC AAC TTG ATT CTG GAC ATT GAT CA - #T GGA AGG CCA ATT AAG          832                                                                          Gln Ala Leu Asn Leu Ile Leu Asp Ile Asp Hi - #s Gly Arg Pro Ile Lys           #           245                                                               - AAT GTA CTA GAC TTA GAG TTT GAT GTG GAA CA - #A CTG AAG GAC TCT ATT          880                                                                          Asn Val Leu Asp Leu Glu Phe Asp Val Glu Gl - #n Leu Lys Asp Ser Ile           #       260                                                                   - GAC AGG GAT AAA ATA GCA GTA ATT GGA CAT TC - #T TTT GGT GGA GCC ACA          928                                                                          Asp Arg Asp Lys Ile Ala Val Ile Gly His Se - #r Phe Gly Gly Ala Thr           #   275                                                                       - GTT CTT CAG GCT CTT AGT GAA GAC CAG AGA TT - #T AGG TGC GGG ATT GCC          976                                                                          Val Leu Gln Ala Leu Ser Glu Asp Gln Arg Ph - #e Arg Cys Gly Ile Ala           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - TTG GAT GCA TGG ATG CTT CCA CTG GAT GAT GC - #A ATA TAT TCC AGA ATC         1024                                                                          Leu Asp Ala Trp Met Leu Pro Leu Asp Asp Al - #a Ile Tyr Ser Arg Ile           #               310                                                           - CCT CAG CCC CTC TTT TTT ATT AAC TCG GAA CG - #G TTC CAA TTT CCT GAG         1072                                                                          Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu Ar - #g Phe Gln Phe Pro Glu           #           325                                                               - AAT ATC AAA AAA ATG AAA AAA TGC TAC TCA CC - #T GAC AAA GAA AGA AAA         1120                                                                          Asn Ile Lys Lys Met Lys Lys Cys Tyr Ser Pr - #o Asp Lys Glu Arg Lys           #       340                                                                   - ATG ATT ACA ATC AGG GGT TCA GTC CAT CAG AA - #C TTT GCT GAT TTC ACT         1168                                                                          Met Ile Thr Ile Arg Gly Ser Val His Gln As - #n Phe Ala Asp Phe Thr           #   355                                                                       - TTT ACA ACT GGC AAA ATA GTT GGA TAC ATA TT - #C ACA TTA AAA GGA GAT         1216                                                                          Phe Thr Thr Gly Lys Ile Val Gly Tyr Ile Ph - #e Thr Leu Lys Gly Asp           360                 3 - #65                 3 - #70                 3 -       #75                                                                           - ATA GAT TCA AAT GTA GCA ATT GAT CTT TGC AA - #C AAA GCT TCA TTG GCA         1264                                                                          Ile Asp Ser Asn Val Ala Ile Asp Leu Cys As - #n Lys Ala Ser Leu Ala           #               390                                                           - TTT TTA CAA AAG CAT TTA GGA CTG CGG AAA GA - #T TTT GAT CAG TGG GAT         1312                                                                          Phe Leu Gln Lys His Leu Gly Leu Arg Lys As - #p Phe Asp Gln Trp Asp           #           405                                                               - TCT TTG ATT GAA GGA AAA GAC GAA AAT CTT AT - #G CCA GGG ACC AAC ATT         1360                                                                          Ser Leu Ile Glu Gly Lys Asp Glu Asn Leu Me - #t Pro Gly Thr Asn Ile           #       420                                                                   - AAC ATC ACC AAC GAA CAT GAC ACT CTA CAG AA - #C TCT CCA GAA GCA GAG         1408                                                                          Asn Ile Thr Asn Glu His Asp Thr Leu Gln As - #n Ser Pro Glu Ala Glu           #   435                                                                       - AAA TCG AAT TTA GAT TAAAAGCACT TTTTTAAAGA TCTTGTTTA - #A AAACTGTCAA         1463                                                                          Lys Ser Asn Leu Asp                                                           440                                                                           - AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAAC TCATATTGGA AA - #ATGTAGGC       1523                                                                          - TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTT TTTCTTTAAA GA - #AAGATTGG       1583                                                                          - TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACT AAAATGATGC TG - #TGTCAAAA       1643                                                                          - TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAAC AGGCACAAGG CT - #AATGAAAA       1703                                                                          - CGTGTTGCAA TGACATAACA ATCCCTAAAA ATACAGATGT TCTTGCCTCT TT - #TTTCTATT       1763                                                                          - ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTT GGAAGCACTT CC - #CTTTGACT       1823                                                                          - TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAA AAGCAGTGTT TT - #ACCAAGGA       1883                                                                          - TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGG GAGTGACCAA GG - #GTAAAAAT       1943                                                                          - ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAA AGAGAGTTTA TA - #ATTTGTTT       2003                                                                          - GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAA GAAAGGTGCG TA - #GGACAGGC       2063                                                                          - CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTT CTTTAATTCT GT - #ATGAGTAT       2123                                                                          - TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAA AAAAAAAAAA AA - #AAAAAAAA       2183                                                                          #        2191                                                                 - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1533 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 62..1394                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CA - #GCTTCGGA         60                                                                          #TGC CTC TGC ACC TGC        106 GCG CTT TTC                                     Met Leu Pro Ser Lys Leu His Ala Leu P - #he Cys Leu Cys Thr Cys             # 15                                                                          - CTT GCA CTG GTT TAT CCT TTT GAC TGG CAA GA - #C CTG AAT CCA GTT GCC          154                                                                          Leu Ala Leu Val Tyr Pro Phe Asp Trp Gln As - #p Leu Asn Pro Val Ala           #                 30                                                          - TAT ATT GAA TCA CCA GCA TGG GTC AGT AAG AT - #A CAA GCT CTG ATG GCT          202                                                                          Tyr Ile Glu Ser Pro Ala Trp Val Ser Lys Il - #e Gln Ala Leu Met Ala           #             45                                                              - GCT GCA AAC ATT GGT CAA TCT AAA ATC CCC AG - #A GGA AAT GGA TCT TAT          250                                                                          Ala Ala Asn Ile Gly Gln Ser Lys Ile Pro Ar - #g Gly Asn Gly Ser Tyr           #         60                                                                  - TCC GTC GGT TGT ACA GAC TTG ATG TTT GAT TA - #C ACT AAT AAG GGC ACC          298                                                                          Ser Val Gly Cys Thr Asp Leu Met Phe Asp Ty - #r Thr Asn Lys Gly Thr           #     75                                                                      - TTC TTG CGT TTG TAT TAT CCA TCT CAA GAT GA - #T GAT CAC TCC GAC ACC          346                                                                          Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp As - #p Asp His Ser Asp Thr           # 95                                                                          - CTT TGG ATC CCA AAC AAA GAA TAT TTT TTG GG - #T CTT AGT AAA TTT CTT          394                                                                          Leu Trp Ile Pro Asn Lys Glu Tyr Phe Leu Gl - #y Leu Ser Lys Phe Leu           #               110                                                           - GGA ACA CAC TGG CTT GTG GGC AAA ATT ATG GG - #C TTA TTC TTC GGT TCA          442                                                                          Gly Thr His Trp Leu Val Gly Lys Ile Met Gl - #y Leu Phe Phe Gly Ser           #           125                                                               - ATG ACA ACT CCT GCA GCC TGG AAT GCA CAT CT - #G AGG ACT GGG GAA AAA          490                                                                          Met Thr Thr Pro Ala Ala Trp Asn Ala His Le - #u Arg Thr Gly Glu Lys           #       140                                                                   - TAC CCA CTA ATT ATT TTT TCT CAT GGT CTT GG - #A GCA TTC AGG ACG ATT          538                                                                          Tyr Pro Leu Ile Ile Phe Ser His Gly Leu Gl - #y Ala Phe Arg Thr Ile           #   155                                                                       - TAT TCT GCT ATT GGC ATT GAT CTG GCA TCC CA - #C GGG TTT ATA GTT GCT          586                                                                          Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser Hi - #s Gly Phe Ile Val Ala           160                 1 - #65                 1 - #70                 1 -       #75                                                                           - GCT GTA GAA CAC AGG GAT GGC TCT GCA TCC TC - #G ACA TAC TAT TTC AAG          634                                                                          Ala Val Glu His Arg Asp Gly Ser Ala Ser Se - #r Thr Tyr Tyr Phe Lys           #               190                                                           - GAC CAG TCT GCT GTA GAA ATA GGC AAC AAG TC - #T TGG CTC TAT CTC AGA          682                                                                          Asp Gln Ser Ala Val Glu Ile Gly Asn Lys Se - #r Trp Leu Tyr Leu Arg           #           205                                                               - ACC CTG AAG CGA GGA GAG GAG GAG TTT CCT TT - #A CGA AAT GAG CAG TTA          730                                                                          Thr Leu Lys Arg Gly Glu Glu Glu Phe Pro Le - #u Arg Asn Glu Gln Leu           #       220                                                                   - CGG CAA CGA GCA AAG GAA TGT TCT CAA GCT CT - #C AGT TTG ATT CTG GAC          778                                                                          Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Le - #u Ser Leu Ile Leu Asp           #   235                                                                       - ATT GAT CAC GGG AGG CCA GTG ACG AAT GTA CT - #A GAT TTA GAG TTT GAT          826                                                                          Ile Asp His Gly Arg Pro Val Thr Asn Val Le - #u Asp Leu Glu Phe Asp           240                 2 - #45                 2 - #50                 2 -       #55                                                                           - GTG GAA CAG CTG AAG GAC TCT ATT GAT AGG GA - #T AAA ATA GCC ATT ATT          874                                                                          Val Glu Gln Leu Lys Asp Ser Ile Asp Arg As - #p Lys Ile Ala Ile Ile           #               270                                                           - GGA CAT TCT TTT GGT GGA GCC ACA GTT ATT CA - #G ACT CTT AGT GAA GAC          922                                                                          Gly His Ser Phe Gly Gly Ala Thr Val Ile Gl - #n Thr Leu Ser Glu Asp           #           285                                                               - CAG AGA TTC AGG TGT GGC ATT GCT CTG GAT GC - #A TGG ATG TTT CCC GTG          970                                                                          Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Al - #a Trp Met Phe Pro Val           #       300                                                                   - GGT GAT GAA GTA TAT TCC AGA ATT CCT CAA CC - #C CTC TTT TTT ATC AAC         1018                                                                          Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pr - #o Leu Phe Phe Ile Asn           #   315                                                                       - TCG GAA CGA TTC CAA TAC CCT TCT AAT ATC AT - #A AGA ATG AAA AAA TGC         1066                                                                          Ser Glu Arg Phe Gln Tyr Pro Ser Asn Ile Il - #e Arg Met Lys Lys Cys           320                 3 - #25                 3 - #30                 3 -       #35                                                                           - TTC TTA CCT GAT AGA GAA CGA AAA ATG ATT AC - #A ATC AGG GGT TCG GTC         1114                                                                          Phe Leu Pro Asp Arg Glu Arg Lys Met Ile Th - #r Ile Arg Gly Ser Val           #               350                                                           - CAT CAG AAT TTT GTT GAC TTC ACT TTT GCC AC - #T AGC AAA ATA ATT GGC         1162                                                                          His Gln Asn Phe Val Asp Phe Thr Phe Ala Th - #r Ser Lys Ile Ile Gly           #           365                                                               - TAC CTA TTC ACA CTG AAA GGA GAC ATC GAT TC - #C AAT GTA GCC ATC AGC         1210                                                                          Tyr Leu Phe Thr Leu Lys Gly Asp Ile Asp Se - #r Asn Val Ala Ile Ser           #       380                                                                   - CTT AGC AAC AAA GCT TCC TTA GCG TTC TTA CA - #A AAA CAT TTA GGA CTT         1258                                                                          Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gl - #n Lys His Leu Gly Leu           #   395                                                                       - CAG AAA GAT TTT GAT CAG TGG GAT TCT TTA GT - #T GAA GGC GAA GAT CAC         1306                                                                          Gln Lys Asp Phe Asp Gln Trp Asp Ser Leu Va - #l Glu Gly Glu Asp His           400                 4 - #05                 4 - #10                 4 -       #15                                                                           - AAT CTT ATT CCA GGG ACC AAC ATT AAC ACA AC - #C AAC CAC CAA GCC ATT         1354                                                                          Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Th - #r Asn His Gln Ala Ile           #               430                                                           - CTG CAG AAC TCC ACA GGA ATA GAG AGA CCA AA - #T TTA GAT T AAAAGAGCTT        1404                                                                          Leu Gln Asn Ser Thr Gly Ile Glu Arg Pro As - #n Leu Asp                       #           440                                                               - TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TT - #AAATGTAT       1464                                                                          - TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA CAAAAAAAAA AA - #AAAAAAAA       1524                                                                          #       1533                                                                  - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1876 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 468..1734                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GC - #CCCTGAGA         60                                                                          - CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CG - #GCCGTGTT        120                                                                          - GGCGCAGCTC CCACGACGTA CGCTTCCTTT CCAGGCTCGA GGAAAGCCTC TC - #CCACAAAC        180                                                                          - ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GC - #GCCCGGCA        240                                                                          - TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CG - #GGACACCG        300                                                                          - CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTTTCCATCC TG - #CCCCCACC        360                                                                          - TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GC - #CGGCTGCT        420                                                                          #GCA TCG      476CGCCGT GGGACCTTCT GCTCTTCCCA ACAAGTG ATG                     #Ala Ser         Met                                                          #1                                                                            - CTG TGG GTG AGA GCC AGG AGG GTG TTC ATG AA - #A AGT CGT GCT TCA GGT          524                                                                          Leu Trp Val Arg Ala Arg Arg Val Phe Met Ly - #s Ser Arg Ala Ser Gly           #      15                                                                     - TTC TCG GCG AAG GCG GCG ACG GAG ATG GGG AG - #C GGC GGC GCG GAG AAG          572                                                                          Phe Ser Ala Lys Ala Ala Thr Glu Met Gly Se - #r Gly Gly Ala Glu Lys           # 35                                                                          - GGC TAT CGG ATC CCC GCC GGG AAG GGC CCG CA - #C GCC GTG GGC TGC ACG          620                                                                          Gly Tyr Arg Ile Pro Ala Gly Lys Gly Pro Hi - #s Ala Val Gly Cys Thr           #                 50                                                          - GAT CTG ATG ACC GGC GAC GCG GCC GAG GGA AG - #C TTT TTG CGC CTG TAT          668                                                                          Asp Leu Met Thr Gly Asp Ala Ala Glu Gly Se - #r Phe Leu Arg Leu Tyr           #             65                                                              - TAC CTA TCG TGT GAC GAC ACA GAT ACT GAA GA - #G ACA CCC TGG ATT CCA          716                                                                          Tyr Leu Ser Cys Asp Asp Thr Asp Thr Glu Gl - #u Thr Pro Trp Ile Pro           #         80                                                                  - GAT AAA GAG TAC TAC CAG GGG CTG TCT GAC TT - #C CTC AAC GTG TAC CGG          764                                                                          Asp Lys Glu Tyr Tyr Gln Gly Leu Ser Asp Ph - #e Leu Asn Val Tyr Arg           #     95                                                                      - GCC CTG GGA GAA AGG CTT TTC CAG TAC TAC GT - #T GGC TCA GTG ACC TGT          812                                                                          Ala Leu Gly Glu Arg Leu Phe Gln Tyr Tyr Va - #l Gly Ser Val Thr Cys           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - CCT GCA AAA TCA AAC GCT GCT TTT AAG CCA GG - #A GAG AAA TAC CCA CTG          860                                                                          Pro Ala Lys Ser Asn Ala Ala Phe Lys Pro Gl - #y Glu Lys Tyr Pro Leu           #               130                                                           - CTC GTT TTT TCC CAT GGA CTT GGA GCT TTT CG - #G ACC ATC TAT TCT GCT          908                                                                          Leu Val Phe Ser His Gly Leu Gly Ala Phe Ar - #g Thr Ile Tyr Ser Ala           #           145                                                               - ATC TGC ATA GAG ATG GCT TCT CAA GGC TTT CT - #A GTG GCA GCT GTG GAG          956                                                                          Ile Cys Ile Glu Met Ala Ser Gln Gly Phe Le - #u Val Ala Ala Val Glu           #       160                                                                   - CAC AGA GAT GAA TCG GCT TCA GCA ACG TAT TT - #C TGT AAA AAG AAG GCT         1004                                                                          His Arg Asp Glu Ser Ala Ser Ala Thr Tyr Ph - #e Cys Lys Lys Lys Ala           #   175                                                                       - GAT TCT GAG CCA GAG GAG GAT CAA ACA TCA GG - #C GTG GAG AAG GAG TGG         1052                                                                          Asp Ser Glu Pro Glu Glu Asp Gln Thr Ser Gl - #y Val Glu Lys Glu Trp           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - ATC TAC TAC AGG AAG CTC AGA GCA GGA GAG GA - #G GAG CGC TGT CTG CGT         1100                                                                          Ile Tyr Tyr Arg Lys Leu Arg Ala Gly Glu Gl - #u Glu Arg Cys Leu Arg           #               210                                                           - CAC AAG CAG GTA CAG CAG AGA GCA CAG GAG TG - #C ATC AAA GCG CTC AAC         1148                                                                          His Lys Gln Val Gln Gln Arg Ala Gln Glu Cy - #s Ile Lys Ala Leu Asn           #           225                                                               - CTC ATT CTT AAG ATC AGT TCA GGA GAG GAA GT - #G ATG AAT GTG CTG AAC         1196                                                                          Leu Ile Leu Lys Ile Ser Ser Gly Glu Glu Va - #l Met Asn Val Leu Asn           #       240                                                                   - TCA GAC TTT GAC TGG AAC CAC CTG AAG GAT TC - #T GTT GAT ACT AGC AGA         1244                                                                          Ser Asp Phe Asp Trp Asn His Leu Lys Asp Se - #r Val Asp Thr Ser Arg           #   255                                                                       - ATA GCT GTG ATG GGA CAC TCT TTT GGT GGT GC - #T ACA GTT ATT GAG AGC         1292                                                                          Ile Ala Val Met Gly His Ser Phe Gly Gly Al - #a Thr Val Ile Glu Ser           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - CTC AGC AAA GAA ATT AGA TTT AGG TGT GGC AT - #T GCC CTT GAT GCG TGG         1340                                                                          Leu Ser Lys Glu Ile Arg Phe Arg Cys Gly Il - #e Ala Leu Asp Ala Trp           #               290                                                           - ATG CTC CCG GTA GGC GAT GAC ACT TAC CAA AG - #C AGT GTG CAG CAA CCA         1388                                                                          Met Leu Pro Val Gly Asp Asp Thr Tyr Gln Se - #r Ser Val Gln Gln Pro           #           305                                                               - CTG CTC TTT ATT AAT TCC GAA AAA TTC CAG TG - #G GCT GCC AAT ATC TTA         1436                                                                          Leu Leu Phe Ile Asn Ser Glu Lys Phe Gln Tr - #p Ala Ala Asn Ile Leu           #       320                                                                   - AAG ATG AAG AAG CTT AGC TCC AAT GAT ACC AA - #C AAG AAA ATG ATC ACC         1484                                                                          Lys Met Lys Lys Leu Ser Ser Asn Asp Thr As - #n Lys Lys Met Ile Thr           #   335                                                                       - ATC AAA GGA TCG GTA CAT CAG AGC TTT CCT GA - #T TTT ACT TTT GTG AGT         1532                                                                          Ile Lys Gly Ser Val His Gln Ser Phe Pro As - #p Phe Thr Phe Val Ser           340                 3 - #45                 3 - #50                 3 -       #55                                                                           - GGA GAA ATC ATT GGA AAG TTT TTC AAG TTA AA - #A GGA GAA ATA GAC CCA         1580                                                                          Gly Glu Ile Ile Gly Lys Phe Phe Lys Leu Ly - #s Gly Glu Ile Asp Pro           #               370                                                           - AAT GAA GCT ATT GAT ATA TGC AAC CAC GCT TC - #A TTG GCC TTC CTG CAG         1628                                                                          Asn Glu Ala Ile Asp Ile Cys Asn His Ala Se - #r Leu Ala Phe Leu Gln           #           385                                                               - AAA CAT CTG AGT CTT AAG AGA GAT TTT GAT AA - #G TGG GAT TCA CTC GTG         1676                                                                          Lys His Leu Ser Leu Lys Arg Asp Phe Asp Ly - #s Trp Asp Ser Leu Val           #       400                                                                   - GAT GGC ATA GGA CCC AAT GTT ATT TCT GGT AC - #C AAT ATC GAC TTA TCT         1724                                                                          Asp Gly Ile Gly Pro Asn Val Ile Ser Gly Th - #r Asn Ile Asp Leu Ser           #   415                                                                       - CCA ACT GAG T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGC - #AGCAGG               1774                                                                          Pro Thr Glu                                                                   420                                                                           - ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CA - #CACAGCTT       1834                                                                          #1876              CAAA AAAAAAAATC ACAGGGGAGC CG                              - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 517 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..514                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #CAA GCC CTA AGT GAA         46 ACA GTT TTT                                     Gly His Ser Phe Gly Gly Ala Thr Val P - #he Gln Ala Leu Ser Glu             # 15                                                                          - GAC CAG AGA TTC AGA TGT GGG ATT GCC CTT GA - #T CCG TGG ATG TTT CCC           94                                                                          Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu As - #p Pro Trp Met Phe Pro           #                 30                                                          - GTG AGT GAG GAG CTG TAC TCC AGA GTT CCT CA - #G CCT CTC TTC TTT ATC          142                                                                          Val Ser Glu Glu Leu Tyr Ser Arg Val Pro Gl - #n Pro Leu Phe Phe Ile           #             45                                                              - AAC TCT GCC GAA TTC CAG ACT CCA AAG GAC AT - #T GCA AAA ATG AAA AAC          190                                                                          Asn Ser Ala Glu Phe Gln Thr Pro Lys Asp Il - #e Ala Lys Met Lys Asn           #         60                                                                  - TTC TAC CAG CCT GAC AAG GAA AGG AAA ATG AT - #T ACG ATC AAG GGC TCA          238                                                                          Phe Tyr Gln Pro Asp Lys Glu Arg Lys Met Il - #e Thr Ile Lys Gly Ser           #     75                                                                      - GTG CAC CAG AAT TTT GCT GAC GGG ACT TTT GT - #A ACT GGC AAA ATA ATT          286                                                                          Val His Gln Asn Phe Ala Asp Gly Thr Phe Va - #l Thr Gly Lys Ile Ile           # 95                                                                          - GGA AAC AAG CTG TCA CTG AAA GGA GAC ATA GA - #C TCC AGA GTT GCC ATA          334                                                                          Gly Asn Lys Leu Ser Leu Lys Gly Asp Ile As - #p Ser Arg Val Ala Ile           #               110                                                           - GAC CTC ACC AAC AAG GCT TCC TTG GCT TTC TT - #A CAA AAA CAT TTA GGA          382                                                                          Asp Leu Thr Asn Lys Ala Ser Leu Ala Phe Le - #u Gln Lys His Leu Gly           #           125                                                               - CTT CAT AAA GAC TTT GAT CAG TGG GAC TGT CT - #G GTG GAG GGA GAG AAC          430                                                                          Leu His Lys Asp Phe Asp Gln Trp Asp Cys Le - #u Val Glu Gly Glu Asn           #       140                                                                   - GAG AAC CTC ATC CCG GGG TCA CCC TTT GAT GT - #A GTC ACC CAG TCC CCG          478                                                                          Glu Asn Leu Ile Pro Gly Ser Pro Phe Asp Va - #l Val Thr Gln Ser Pro           #   155                                                                       #    517G CAG AGT TCT CCC GGA TCA CAC AAC CA - #G AAT TAG                     Ala Leu Gln Ser Ser Pro Gly Ser His Asn Gl - #n Asn                           160                 1 - #65                 1 - #70                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 580 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..580                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GA - #A CGT AAA ATC CCT AAG           48                                                                          Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gl - #u Arg Lys Ile Pro Lys           #                 15                                                          - GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA GA - #C TTA ATG TTT GAT TAC           96                                                                          Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr As - #p Leu Met Phe Asp Tyr           #             30                                                              - ACT AAA AAG GGC ACC TTC TTG CGT TTA TAT TA - #T CCA TCC CAA GAT GAT          144                                                                          Thr Lys Lys Gly Thr Phe Leu Arg Leu Tyr Ty - #r Pro Ser Gln Asp Asp           #         45                                                                  - GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT AA - #G GAG TAT TTT TGG GGT          192                                                                          Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Ly - #s Glu Tyr Phe Trp Gly           #     60                                                                      - CTT AGC AAG TAT CTT GGA AAA CAC TGG CTT AT - #G GGC AAC ATT TTG AGT          240                                                                          Leu Ser Lys Tyr Leu Gly Lys His Trp Leu Me - #t Gly Asn Ile Leu Ser           # 80                                                                          - TTA CTC TTT GGT TCA GTG ACA ACT CCT GCA AA - #C TGG AAT TCC CCT CTG          288                                                                          Leu Leu Phe Gly Ser Val Thr Thr Pro Ala As - #n Trp Asn Ser Pro Leu           #                 95                                                          - AGG CCT GGT GAA AAA TAC CCA CTT GTT GTT TT - #T TCT CAT GGT CTT GGA          336                                                                          Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Ph - #e Ser His Gly Leu Gly           #           110                                                               - GCA TTC AGG ACA ATT TAT TCT GCT ATT GGC AT - #T GAC CTG GCA TCT CAT          384                                                                          Ala Phe Arg Thr Ile Tyr Ser Ala Ile Gly Il - #e Asp Leu Ala Ser His           #       125                                                                   - GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA GA - #T AGA TCT GCA TCT GCA          432                                                                          Gly Phe Ile Val Ala Ala Val Glu His Arg As - #p Arg Ser Ala Ser Ala           #   140                                                                       - ACT TAC TAT TTC AAG AAC CAA TCT GCT GCA GA - #A ATA GGG AAA AAG TCT          480                                                                          Thr Tyr Tyr Phe Lys Asn Gln Ser Ala Ala Gl - #u Ile Gly Lys Lys Ser           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - TGG CTC TAC CTT AGA ACC CTG AAA GAA GAG GA - #G GAG ATA CAT ATA CGA          528                                                                          Trp Leu Tyr Leu Arg Thr Leu Lys Glu Glu Gl - #u Glu Ile His Ile Arg           #               175                                                           - AAT AAG CAG GTA CGA CAA AGA GCA AAA GAA TG - #T TCC CAA GCT CTC AGT          576                                                                          Asn Lys Gln Val Arg Gln Arg Ala Lys Glu Cy - #s Ser Gln Ala Leu Ser           #           190                                                               #            580                                                              Leu                                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Gly Xaa Ser Xaa Gly                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #   41             TACA AGTATTAATG GCTGCTGCAA G                               - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #          32      TATT TCTCTATTCC TG                                         - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1335 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CG - #TCGTGTAC         60                                                                          - CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CT - #GGGTGAAT        120                                                                          - AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TA - #GAGGCAAC        180                                                                          - GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AG - #GAACTTTT        240                                                                          - CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GA - #TCCCAAAT        300                                                                          - AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GG - #GCAACATT        360                                                                          - TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TC - #TGAGGCCT        420                                                                          - GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GA - #CACTTTAT        480                                                                          - TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AG - #AACACAGA        540                                                                          - GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AA - #TAGGGGAC        600                                                                          - AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT AC - #GAAATGAG        660                                                                          - CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TG - #ACATTGAT        720                                                                          - CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA AC - #TGAAGGAC        780                                                                          - TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AA - #CGGTTATT        840                                                                          - CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC AT - #GGATGTTT        900                                                                          - CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT CA - #ACTCTGAA        960                                                                          - TATTTCCAAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC TG - #ATAAAGAA       1020                                                                          - AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT CA - #CTTTTGCA       1080                                                                          - ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC AA - #ATGTAGCT       1140                                                                          - ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG AC - #TTCATAAA       1200                                                                          - GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT TC - #CAGGGACC       1260                                                                          - AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT AG - #AGAAATAC       1320                                                                          #  1335                                                                       __________________________________________________________________________

We claim:
 1. A method of treating a mammal susceptible to or sufferingfrom pre-term labor comprising administering a pharmaceuticalcomposition comprising platelet-activating factor acetyl hydrolase(PAF-AH) enzyme to said mammal in an amount sufficient to supplementendogenous PAF-AH activity and to inactivate pathological amounts ofPAF, wherein said PAF-AH enzyme comprises the mature human amino acidsequence of SEQ ID NO: 8 or comprises a PAF-AH polypeptide fragment,variant or variant fragment that hydrolyzes ³ H-acetate from PAF, saidPAF-AH enzyme being produced by growing a host cell transformed ortransfected with a DNA and isolating said PAF-AH enzyme from said hostcell or the medium of its growth, said DNA being selected from the groupconsisting of (a) a DNA comprising the sequence set out in SEQ ID NO: 7,and (b) a DNA which hybridizes under stringent conditions to thenon-coding strand of (a).
 2. A method of treating a mammal susceptibleto or suffering from pre-term labor comprising administering apharmaceutical composition comprising platelet-activating factor acetylhydrolase (PAF-AH) enzyme to said mammal in an amount sufficient tosupplement endogenous PAF-AH activity and to inactivate pathologicalamounts of PAF, wherein said PAF-AH enzyme comprises the mature humanamino acid sequence of SEQ ID NO: 8 or comprises a PAF-AH polypeptidefragment, variant or variant fragment that hydrolyzes ³ H-acetate fromPAF, said PAF-AH enzyme being encoded by a DNA selected from the groupconsisting of (a) a DNA comprising the sequence set out in SEQ ID NO: 7,and (b) a DNA which hybridizes under stringent conditions to thenon-coding strand of (a).